CN112898437B - New coronavirus antigen and preparation method and application thereof - Google Patents

New coronavirus antigen and preparation method and application thereof Download PDF

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CN112898437B
CN112898437B CN202110312107.XA CN202110312107A CN112898437B CN 112898437 B CN112898437 B CN 112898437B CN 202110312107 A CN202110312107 A CN 202110312107A CN 112898437 B CN112898437 B CN 112898437B
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CN112898437A (en
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任宝永
刘鹏
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Sige Suzhou Biotechnology Co ltd
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Abstract

A novel coronavirus antigen comprises a fusion protein which comprises a Spike protein S1 domain part and an HSA part, wherein arginine at 685 position corresponding to the Spike protein in the Spike protein S1 domain part is mutated into alanine. The fusion protein prepared by the invention is used as a key antigen component in a new coronavirus serum antibody detection kit and is used for detecting a new coronavirus serum antibody; antibodies are produced by administration to animals or humans as vaccines for the prevention of novel coronaviruses. The new coronavirus pneumonia is treated by administering the new coronavirus as a therapeutic drug to a new coronapatient.

Description

New coronavirus antigen and preparation method and application thereof
Technical Field
The invention belongs to the fields of biology and medicine, and relates to a novel coronavirus antigen, a preparation method and application thereof.
Background
The novel coronavirus (SARS-CoV-2) belongs to the class of beta coronaviruses (Betacoronavirus) and is a single-stranded positive-strand RNA virus. Adjacent to the SARS virus and class of SARS (SARS-like) virus at the position of the evolutionary tree. The disease is mainly manifested by fever, dry cough and hypodynamia, often accompanied by dyspnea and dyspnea, and in severe cases, the new coronaries pneumonia can lead to severe acute respiratory distress syndrome, sepsis shock, multiple functions failure and even death. Coronaviruses structurally mainly comprise Spike (S), envelope (Envelope), nucleoprotein (N) and Envelope (M). The S protein forms a special corolla structure on the surface of the virus in a trimer form, and is split into two subunits S1 and S2 under the action of host protease, wherein the S1 subunit comprises a Receptor Binding Domain (RBD), the main function is binding to a host cell surface receptor, and the S2 subunit mainly functions to mediate the membrane fusion process of the virus and the cell, the key characteristics of the S protein comprise fusion peptide, two heptapeptide repeat domains (called HR1 and HR 2) and a transmembrane domain, and the interaction of the S protein trimer and the transmembrane domain is considered to be the key for completing the membrane fusion process of the virus infection.
Nucleic acid detection is a gold standard for the current diagnosis of new coronaries, but the nucleic acid detection has limitations, such as sensitivity problem of the reagent itself, missed detection may occur in the nucleic acid detection, and some infected persons have low virus content at the nasal mucosa sampling position at the initial stage of infection, and the virus carrier cannot be found in time due to the fact that the nucleic acid detection is negative after multiple times. After infection of the human body by the virus, the immune system of the body produces antibodies specific for the virus, including IgM and IgG. By detecting the virus specific antibody in serum, the virus carrier is timely judged and found. Therefore, antibody detection is an important supplement to nucleic acid detection and has important value. The most important reagent component for antibody detection is antigen, and the stability of the antigen and the biological activity of the antigen determine the sensitivity of the detection reagent.
Most antigens used in the current common novel coronavirus antibody detection kit on the market are RBD domains aiming at Spike proteins, the NTD domains are highly glycosylated parts, and compared with RBD segments, the antigen has stronger immunogenicity and is easier to excite antibodies generated in vivo, so that the RBD domains serving as anti-principle cannot effectively detect the antibodies aiming at the NTD, and the sensitivity of detection reagents is reduced.
Disclosure of Invention
The invention aims to provide a novel coronavirus antigen and a preparation method and application thereof.
To achieve the above and other related objects, the present invention provides the following technical solutions: a fusion protein comprising a Spike protein S1 domain portion and an HSA portion, wherein an arginine corresponding to position 685 of the Spike protein in the Spike protein S1 domain portion is mutated to an alanine.
The preferable technical scheme is as follows: the fusion protein has the structure of S1-HSA or HSA-S1, wherein "-" represents a chemical bond or a linker.
The preferable technical scheme is as follows: the Spike eggThe amino acid sequence of the white S1 domain part is SEQ ID No ： 1。
The preferable technical scheme is as follows: the amino acid sequence of the fusion protein is SEQ ID No ： 2。
To achieve the above and other related objects, the present invention provides the following technical solutions: a nucleotide encoding a fusion protein, the nucleotide having the sequence of SEQ ID No:3.
to achieve the above and other related objects, the present invention provides the following technical solutions: a recombinant vector comprising the nucleotide described above.
To achieve the above and other related objects, the present invention provides the following technical solutions: an expression cassette comprising the nucleotide described above.
To achieve the above and other related objects, the present invention provides the following technical solutions: a microorganism or host cell comprising the above nucleotide.
To achieve the above and other related objects, the present invention provides the following technical solutions: a pharmaceutical composition comprising the fusion protein described above.
To achieve the above and other related objects, the present invention provides the following technical solutions: the fusion protein is used as antigen in preparing new coronavirus detection reagent.
Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:
the fusion protein prepared by the invention is used as a key antigen component in a new coronavirus serum antibody detection kit and is used for detecting a new coronavirus serum antibody; antibodies are produced by administration to animals or humans as vaccines for the prevention of novel coronaviruses. The new coronavirus pneumonia is treated by administering the new coronavirus as a therapeutic drug to a new coronapatient.
Drawings
FIG. 1 shows a recombinant plasmid pCDNA3.1-S1-HSA map of the recombinant S1-HSA fusion protein provided in the examples of the present invention.
FIG. 2 shows SDS-PAGE patterns of molecular weight detection of recombinant S1-HSA fusion proteins provided in examples of the present invention.
FIG. 3 is a graph showing the comparison of the expression levels of recombinant S1-HSA fusion protein and unmutated S1 (WT) -HSA recombinant protein provided in the examples of the present invention.
FIG. 4 shows an ELISA binding experiment of recombinant S1-HSA fusion protein and ACE2 molecule provided by the embodiment of the invention.
FIG. 5 shows the binding test of recombinant S1-HSA fusion proteins provided in the examples of the present invention to RBD monoclonal antibodies and NTD monoclonal antibodies.
Detailed Description
Further advantages and effects of the present invention will become apparent to those skilled in the art from the disclosure of the present invention, which is described by the following specific examples.
Please refer to fig. 1-5. It should be understood that the structures, proportions, sizes, etc. shown in the drawings are shown only in the drawings and should not be taken as limiting the invention to those having ordinary skill in the art, since modifications, changes in proportions, or adjustments of sizes, etc. could be made without departing from the spirit or essential characteristics of the invention. The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the examples described below were purchased from conventional biochemical reagent stores unless otherwise specified.
Example 1: new coronavirus antigen and preparation method and application thereof
Preparation of fusion protein S1-HSA:
spike gene was purchased from Nanjing Jinsri Biotechnology Co., ltd (cat# C0425FA 280-6), and HSA gene was prepared by gene synthesis by Soujin Zhi Bio Inc. The synthetic primers were designed as follows:
S-F0:ACCCAAGCTTgccaccATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGTTCCTCACAGTGCGTCAATCTG；
S1-R1: CCTCGCTCTTGTGGGCGTCACCTCCTGGCGCGCCCGCGGCTCTTCTGGGAGAGTTTG；
HSA-F1: CCAGAAGAGCCGCGGGCGCGCCAGGAGGTGACGCCCACAAGAGCGAGGTGGCCC；
HSA-R2: ATCTGCAGAATTCCCAGGCCCAGGGCGGCCTGGCTG；
the Spike gene is used as a template, S-F0/S1-R1 is used as a primer, S1 gene fragments are amplified, and amplification conditions are as follows: 94 ℃ for 5min; (94 ℃,30s;55 ℃,30s;72 ℃,1 min) x30;72℃for 5min. Amplification system: 10Xbuffer (containing Mg 2+ ) 5ul;2.5mM dNTP,2ul;10uMprimer1,1ul;10uM primer2,1ul; template, 2ul; taq,0.5ul; ddH 2 O,38.5ul. HSA gene is used as a template, HSA-F1/HSA-R2 is used as a primer, an HSA fragment is amplified, and amplification conditions are as follows: 94 ℃ for 5min; (94 ℃,30s;55 ℃,30s;72 ℃,1 min) x30;72℃for 5min. Amplification system: 10 Xbaffer (containing Mg 2+ ) 5ul;2.5mM dNTP,2ul;10uMprimer1,1ul;10uM primer2,1ul; template, 2ul; taq,0.5ul; ddH 2 O,38.5ul. And then the amplified S1 fragment and the amplified HSA fragment are used as templates, S-F0 and HSA-R2 are used as primers, the S1-HSA fusion fragment is amplified, and the amplification conditions are as follows: 94 ℃ for 5min; (94 ℃,30s;55 ℃,30s;72 ℃,1 min) x30;72℃for 5min. Amplification system: 10Xbuffer (containing Mg 2+ ) 5ul;2.5mM dNTP,2ul;10uM primer1,1ul;10uM primer2,1ul; template, 2ul; taq,0.5ul; ddH 2 O，38.5ul。
The PCDNA3.1 plasmid vector was digested with HindIII/EcoRI and the large fragment was recovered using a gel recovery kit purchased from Tiangen Biochemical, cat: DP209-03, see the description for the procedure. The S1-HSA PCR product was digested with HindIII/EcoRI, the digestion system: buffer 2X: 2ul, ecoRI:0.5ul, hindIII:0.5ul, template PCR product 2ul, water: 15ul,37 ℃ for 30 minutes, then recovered using a glue recovery kit purchased from the root biochemistry, cat No.: DP209-03, see the description for the procedure. The vector and fragment recovered by digestion were ligated for 30 min with T4 DNase (ThermoFisher, cat# 46300018) and the ligated product was transformed into TOP10 E.coli competent, incubated overnight at 37℃and the next day was picked for clone sequencing identification. For the correct sequencing clones, the recombinant plasmid in E.coli TOP10 was extracted with endotoxin-free plasmid extraction kit, designated: pCDNA3.1-S1-HSA.
To construct the pCDNA3.1-S1 (wt) -HSA plasmid, the synthetic primers were designed as follows:
S-F0:ACCCAAGCTTgccaccATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGTTCCTCACAGTGCGTCAATCTG；
S1-R1’：
CCTCGCTCTTGTGGGCGTCACCTCCTGGCGCGCCtctGGCTCTTCTGGGAGAGTTTG；
HSA-F1’：
CCAGAAGAGCCagaGGCGCGCCAGGAGGTGACGCCCACAAGAGCGAGGTGGCCC；
HSA-R2’：ATCTGCAGAATTCCCAGGCCCAGGGCGGCCTGGCTG；
s1 (WT) gene fragment was amplified using Spike gene as template and S-F0/S1-R' as primer, amplification conditions: 94 ℃ for 5min; (94 ℃,30s;55 ℃,30s;72 ℃,1 min) x30;72℃for 5min. Amplification system: 10Xbuffer (containing Mg2+), 5ul;2.5mM dNTP,2ul;10uMprimer1,1ul;10uM primer2,1ul; template, 2ul; taq,0.5ul; ddH 2 O,38.5ul. HSA gene is used as a template, HSA-F1 '/HSA-R2' is used as a primer, an HSA fragment is amplified, and amplification conditions are as follows: 94 ℃ for 5min; (94 ℃,30s;55 ℃,30s;72 ℃,1 min) x30;72℃for 5min. Amplification system: 10Xbuffer (containing Mg2+), 5ul;2.5mM dNTP,2ul;10uMprimer1,1ul;10uM primer2,1ul; template, 2ul; taq,0.5ul; ddH 2 O,38.5ul. And then using the amplified S1 (WT) fragment and HSA fragment as templates, and S-F0 and HSA-R2' as primers to amplify the S1-HSA fusion fragment. The PCDNA3.1 plasmid vector was digested with HindIII/EcoRI and the large fragment was recovered by gel, the S1-HSA PCR product was digested with HindIII/EcoRI and gel was recovered, the digested and recovered vector and fragment were ligated with T4 DNase (ThermoFisher, cat# 46300018) for 30 minutes, the ligated product was transformed into TOP10 E.coli competent, cultured overnight at 37℃and the next day was picked for clone sequencing identification. For the correct sequencing clones, the recombinant plasmid in E.coli TOP10 was extracted with endotoxin-free plasmid extraction kit, designated: pCDNA3.1-S1 (wt) -HSA.
293F cells were cultured until the cell density reached 2X 10 6 –3×10 6 Cell/ml was subjected to reconstitutionTransfection of group plasmid (transfection reagent: thermo, cat# A14525), culturing for 7 days after transfection, collecting supernatant, high-speed centrifugation, purification of fusion protein by HSA affinity chromatography (chromatography packing: thermo, cat# 191297050).
FIG. 2 is a SDS-PAGE chart showing the molecular weight measurement of recombinant S1-HSA fusion protein, and the molecular weight of the purified fusion protein was measured by SDS-PAGE. As can be seen from FIG. 2, the recombinant expression vector for expressing the fusion protein S1-HSA has been successfully constructed, and the expression and purification of the fusion protein S1-HSA in host cells has been achieved.
Comparison of protein expression level: 53mg/L of S1-HSA, 22mg/L of S1 (wt) -HSA and 685 th arginine (Arg) of S1 sequence are mutated into alanine (Ala), so that the expression level of the fusion protein is unexpectedly improved, and the production cost can be obviously reduced in later application, as shown in figure 3.
Detection of the action of fusion protein S1-HSA with ACE2 protein:
the novel coronavirus Spike protein can interact with human ACE2 protein under the condition of having correct spatial conformation, and in order to verify whether the expressed recombinant protein S1-HSA has correct conformation and biological activity, the combination experiment of the novel coronavirus Spike protein and ELISA of ACE2 is used for verification.
And (3) preparation of a reagent:
(1) coating liquid: 0.05mol/L carbonate buffer (pH 9.6)
0.75g of sodium carbonate, 1.46g of sodium bicarbonate and deionized water were added to a volume of 500ml.
(2) 0.02mol/L phosphate buffer (pH 7.4)
0.2g of monopotassium phosphate, 2.90g of disodium phosphate, 8g of sodium chloride and deionized water are added to a volume of 1000ml.
(3) Antibody dilution: 0.02mol/L PBS (pH 7.4) +0.2% BSA
0.2g of BSA was dissolved in the formulated phosphate buffer of 0.02mol/L and quantified to 100g.
(4) Sealing liquid: 0.05mol/L carbonate buffer (pH 9.6) +2.0% BSA
2.0g BSA was dissolved in 0.05mol/L carbonate buffer and quantified to 100g.
(5) Washing liquid: 0.02mol/L PBS (pH 7.4) +0.05% Tween-20
50ul Tween-20 was dissolved in 100ml of 0.02mol/L phosphate buffer solution, and mixed by shaking.
(6) Color development liquid: TMB-hydrogen peroxide urea solution
Solution A (3, 3', 5' -tetramethylbenzidine, TMB) was prepared by weighing 20mg of TMB, dissolving in 10ml of absolute ethanol, and adding double distilled water to 100ml.
Solution B (0.1 mol/L citric acid-0.2 mol/L disodium hydrogen phosphate buffer solution, pH 5.0-5.4) is prepared by weighing 14.34g of Na2HPO4.12H2O, dissolving 1.87g of citric acid in 180ml double distilled water, adding 1.28ml of 0.75% hydrogen peroxide urea, fixing the volume to 200ml, and adjusting pH to 5.0-5.4.
Mixing the solution A and the solution B according to the proportion of 1: and mixing to obtain TMB-hydrogen peroxide urea application liquid.
(7) Stop solution: 2mol/L H 2 SO 4 Solution
10ml of 98% concentrated sulfuric acid is added into 60ml of double distilled water, the volume is fixed to 100ml, and the mixture is stored at room temperature.
(8) Enzyme-labeled secondary antibody: HRP-labeled goat anti-mouse IgG was diluted 3000-fold with antibody diluent at the time of application.
The experimental method comprises the following steps:
antigen coating
The antigen S1-HSA was diluted to 0.01mg/ml with a coating solution (0.05 mol/L carbonate buffer, pH 9.6), and 100. Mu.l of the diluted antigen solution was added to each ELISA plate well. The ELISA plate was placed in a wet box and coated overnight at 4 ℃.
(2) Washing plate
The coating liquid is discarded, all enzyme-labeled holes are filled with the washing liquid, and the buckle is covered by gauze and toilet paper. And (3) buckling the water for 1 time and drying the water last time.
(3) Closure
Mu.l of blocking solution (pH 9.6, 0.05mol/L carbonate buffer containing 2.0% BSA) was added to each well of the plate, and the plate was placed in a wet box and incubated overnight at 4 ℃. Incubation may also be carried out for 2h at 37 ℃.
(4) The blocking solution was discarded and washed 1 time as above.
(5) Add first antibody (ACE 2-mFC)
ACE2-mFC. The maximum concentration was set at 10ug/ml, dilutions were made at 3-fold gradients, 11 total and one 0 concentration spot, 100ul was added per well, and after addition the plates were incubated in a wet box at 37℃for 1h.
(6) Washing is the same as above.
(7) Enzyme-labeled secondary antibody
100 μl of enzyme-labeled secondary antibody (goat anti-mouse) with dilution of 1:10000 was added to each well, and incubated in a 37℃wet box for 1h.
(8) Washing and installing
(9) Color development
After 50 μl of solution A and solution B are added to each well, the ELISA plate is placed in a wet box and protected from light for about 3min, and the ELISA plate is terminated when a negative control Kong Xian is blue-green. At the end, 50. Mu.l of 2mol/L concentrated sulfuric acid was added to each well.
(10) Detection of
After termination, the value of each well A450 of the ELISA plate was measured rapidly.
The experimental results show that S1-HSA can be obviously combined with ACE2 protein, and the S1-HSA recombinant protein has complete spatial conformation and biological activity, as shown in figure 4.
Effect detection of fusion protein S1-HSA with novel coronavirus detection antibody:
the S1 domain in the novel coronavirus Spike protein also comprises an NTD domain and an RBD domain, and in order to verify whether the S1-HSA recombinant protein has the complete NTD domain and RBD domain, detection is performed by the NTD domain and the RBD specific antibody. ELISA experimental procedure the same as in example 2 was followed by coating the S1-HSA antigen, ELISA detection with NTD domain-specific antibodies and RBD-specific antibodies, the highest concentration of antibodies was 10ug/ml, 2-fold concentration dilution, 11 concentration spots and 1 concentration spot of 0 concentration.
The experimental results show that: the S1-HSA recombinant protein can be effectively identified by NTD antibodies and RBD antibodies, has complete biological conformation, and can be used for detecting clinical blood freshening crown antibodies.
Example 2: new coronavirus antigen and preparation method and application thereof
A fusion protein comprising a Spike protein S1 domain portion and an HSA portion, wherein an arginine corresponding to position 685 of the Spike protein in the Spike protein S1 domain portion is mutated to an alanine.
The preferable technical scheme is as follows: the fusion protein has the structure of S1-HSA or HSA-S1, wherein "-" represents a chemical bond or a linker.
The preferable technical scheme is as follows: the amino acid sequence of the Spike protein S1 structural domain part is SEQ ID No ： 1。
The preferable technical scheme is as follows: the amino acid sequence of the fusion protein is SEQ ID No ： 2。
To achieve the above and other related objects, the present invention provides the following technical solutions: a nucleotide encoding a fusion protein, the nucleotide having the sequence of SEQ ID No:3.
a recombinant vector comprising the nucleotide described above.
An expression cassette comprising the nucleotide described above.
A microorganism or host cell comprising the above nucleotide.
A pharmaceutical composition comprising the fusion protein described above.
The fusion protein is used as antigen in preparing new coronavirus detection reagent.
The foregoing description of the preferred embodiment of the invention is not intended to be limiting in any way, but rather, it is intended to cover all modifications or variations of the invention which fall within the spirit and scope of the invention.
Sequence listing
<110> Siger (Suzhou) Biotech Co., ltd
<120> a novel coronavirus antigen, and preparation method and application thereof
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<170> PatentIn version 3.5
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<213> amino acid sequence of Spike protein S1 Domain
<400> 1
Ser Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala
1 5 10 15
Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe
20 25 30
Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe
35 40 45
Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly
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Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr
65 70 75 80
Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly
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Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala
100 105 110
Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro
115 120 125
Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser
130 135 140
Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val
145 150 155 160
Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys
165 170 175
Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile
180 185 190
Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly
195 200 205
Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile
210 215 220
Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro
225 230 235 240
Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val
245 250 255
Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly
260 265 270
Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr
275 280 285
Lys Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr
290 295 300
Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn
305 310 315 320
Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe
325 330 335
Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala
340 345 350
Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys
355 360 365
Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val
370 375 380
Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala
385 390 395 400
Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp
405 410 415
Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser
420 425 430
Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser
435 440 445
Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala
450 455 460
Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro
465 470 475 480
Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro
485 490 495
Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr
500 505 510
Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val
515 520 525
Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser
530 535 540
Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp
545 550 555 560
Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile
565 570 575
Thr Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn
580 585 590
Thr Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu
595 600 605
Val Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val
610 615 620
Tyr Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile
625 630 635 640
Gly Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly
645 650 655
Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg
660 665 670
Ala Ala
<210> 2
<211> 1278
<212> PRT
<213> amino acid sequence of fusion protein
<400> 2
Ser Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala
1 5 10 15
Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe
20 25 30
Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe
35 40 45
Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly
50 55 60
Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr
65 70 75 80
Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly
85 90 95
Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala
100 105 110
Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro
115 120 125
Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser
130 135 140
Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val
145 150 155 160
Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys
165 170 175
Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile
180 185 190
Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly
195 200 205
Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile
210 215 220
Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro
225 230 235 240
Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val
245 250 255
Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly
260 265 270
Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr
275 280 285
Lys Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr
290 295 300
Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn
305 310 315 320
Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe
325 330 335
Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala
340 345 350
Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys
355 360 365
Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val
370 375 380
Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala
385 390 395 400
Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp
405 410 415
Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser
420 425 430
Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser
435 440 445
Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala
450 455 460
Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro
465 470 475 480
Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro
485 490 495
Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr
500 505 510
Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val
515 520 525
Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser
530 535 540
Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp
545 550 555 560
Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile
565 570 575
Thr Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn
580 585 590
Thr Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu
595 600 605
Val Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val
610 615 620
Tyr Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile
625 630 635 640
Gly Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly
645 650 655
Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg
660 665 670
Ala Ala Gly Ala Pro Gly Gly Asp Ala His Lys Ser Glu Val Ala His
675 680 685
Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile
690 695 700
Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys
705 710 715 720
Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu
725 730 735
Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys
740 745 750
Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp
755 760 765
Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His
770 775 780
Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp
785 790 795 800
Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys
805 810 815
Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu
820 825 830
Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys
835 840 845
Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu
850 855 860
Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala
865 870 875 880
Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala
885 890 895
Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys
900 905 910
Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp
915 920 925
Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys
930 935 940
Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys
945 950 955 960
Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu
965 970 975
Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys
980 985 990
Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met
995 1000 1005
Phe Leu Tyr Glu Tyr Ala Arg Arg His ProAsp Tyr Ser Val Val
1010 1015 1020
Leu Leu Leu Arg Leu Ala Lys Thr Tyr GluThr Thr Leu Glu Lys
1025 1030 1035
Cys Cys Ala Ala Ala Asp Pro His Glu CysTyr Ala Lys Val Phe
1040 1045 1050
Asp Glu Phe Lys Pro Leu Val Glu Glu ProGln Asn Leu Ile Lys
1055 1060 1065
Gln Asn Cys Glu Leu Phe Glu Gln Leu GlyGlu Tyr Lys Phe Gln
1070 1075 1080
Asn Ala Leu Leu Val Arg Tyr Thr Lys LysVal Pro Gln Val Ser
1085 1090 1095
Thr Pro Thr Leu Val Glu Val Ser Arg AsnLeu Gly Lys Val Gly
1100 1105 1110
Ser Lys Cys Cys Lys His Pro Glu Ala LysArg Met Pro Cys Ala
1115 1120 1125
Glu Asp Tyr Leu Ser Val Val Leu Asn GlnLeu Cys Val Leu His
1130 1135 1140
Glu Lys Thr Pro Val Ser Asp Arg Val ThrLys Cys Cys Thr Glu
1145 1150 1155
Ser Leu Val Asn Arg Arg Pro Cys Phe SerAla Leu Glu Val Asp
1160 1165 1170
Glu Thr Tyr Val Pro Lys Glu Phe Asn AlaGlu Thr Phe Thr Phe
1175 1180 1185
His Ala Asp Ile Cys Thr Leu Ser Glu LysGlu Arg Gln Ile Lys
1190 1195 1200
Lys Gln Thr Ala Leu Val Glu Leu Val LysHis Lys Pro Lys Ala
1205 1210 1215
Thr Lys Glu Gln Leu Lys Ala Val Met AspAsp Phe Ala Ala Phe
1220 1225 1230
Val Glu Lys Cys Cys Lys Ala Asp Asp LysGlu Thr Cys Phe Ala
1235 1240 1245
Glu Glu Gly Lys Lys Leu Val Ala Ala SerGln Ala Ala Leu Gly
1250 1255 1260
Leu Gly Ile Leu Gln Ile Ser Ser Thr ValAla Ala Ala Arg Val
1265 1270 1275
<210> 3
<211> 3834
<212> DNA
<213> nucleotide sequence encoding fusion protein
<400> 3
tcctcacagt gcgtcaatct gacaactcgg actcagctgc cacctgctta tactaatagc 60
ttcaccagag gcgtgtacta tcctgacaag gtgtttagaa gctccgtgct gcactctaca 120
caggatctgt ttctgccatt ctttagcaac gtgacctggt tccacgccat ccacgtgagc 180
ggcaccaatg gcacaaagcg gttcgacaat cccgtgctgc cttttaacga tggcgtgtac 240
ttcgcctcta ccgagaagag caacatcatc agaggatgga tctttggcac cacactggac 300
tccaagacac agtctctgct gatcgtgaac aatgccacca acgtggtcat caaggtgtgc 360
gagttccagt tttgtaatga tcccttcctg ggcgtgtact atcacaagaa caataagagc 420
tggatggagt ccgagtttag agtgtattct agcgccaaca actgcacatt tgagtacgtg 480
agccagcctt tcctgatgga cctggagggc aagcagggca atttcaagaa cctgagggag 540
ttcgtgttta agaatatcga cggctacttc aaaatctact ctaagcacac ccccatcaac 600
ctggtgcgcg acctgcctca gggcttcagc gccctggagc ccctggtgga tctgcctatc 660
ggcatcaaca tcacccggtt tcagacactg ctggccctgc acagaagcta cctgacaccc 720
ggcgactcct ctagcggatg gaccgccggc gctgccgcct actatgtggg ctacctccag 780
ccccggacct tcctgctgaa gtacaacgag aatggcacca tcacagacgc agtggattgc 840
gccctggacc ccctgagcga gacaaagtgt acactgaagt cctttaccgt ggagaagggc 900
atctatcaga catccaattt cagggtgcag ccaaccgagt ctatcgtgcg ctttcctaat 960
atcacaaacc tgtgcccatt tggcgaggtg ttcaacgcaa cccgcttcgc cagcgtgtac 1020
gcctggaata ggaagcggat cagcaactgc gtggccgact atagcgtgct gtacaactcc 1080
gcctctttca gcacctttaa gtgctatggc gtgtccccca caaagctgaa tgacctgtgc 1140
tttaccaacg tctacgccga ttctttcgtg atcaggggcg acgaggtgcg ccagatcgcc 1200
cccggccaga caggcaagat cgcagactac aattataagc tgccagacga tttcaccggc 1260
tgcgtgatcg cctggaacag caacaatctg gattccaaag tgggcggcaa ctacaattat 1320
ctgtaccggc tgtttagaaa gagcaatctg aagcccttcg agagggacat ctctacagaa 1380
atctaccagg ccggcagcac cccttgcaat ggcgtggagg gctttaactg ttatttccca 1440
ctccagtcct acggcttcca gcccacaaac ggcgtgggct atcagcctta ccgcgtggtg 1500
gtgctgagct ttgagctgct gcacgcccca gcaacagtgt gcggccccaa gaagtccacc 1560
aatctggtga agaacaagtg cgtgaacttc aacttcaacg gcctgaccgg cacaggcgtg 1620
ctgaccgagt ccaacaagaa gttcctgcca tttcagcagt tcggcaggga catcgcagat 1680
accacagacg ccgtgcgcga cccacagacc ctggagatcc tggacatcac accctgctct 1740
ttcggcggcg tgagcgtgat cacacccggc accaatacaa gcaaccaggt ggccgtgctg 1800
tatcaggacg tgaattgtac cgaggtgccc gtggctatcc acgccgatca gctgacccca 1860
acatggcggg tgtacagcac cggctccaac gtcttccaga caagagccgg atgcctgatc 1920
ggagcagagc acgtgaacaa ttcctatgag tgcgacatcc caatcggcgc cggcatctgt 1980
gcctcttacc agacccagac aaactctccc agaagagccg cgggcgcgcc aggaggtgac 2040
gcccacaaga gcgaggtggc ccaccgcttc aaggacctgg gcgaggagaa cttcaaggcc 2100
ctggtgctga tcgccttcgc ccagtacctg cagcagtgcc ccttcgagga ccacgtgaag 2160
ctggtgaacg aggtgaccga gttcgccaag acctgcgtgg ccgacgagag cgccgagaac 2220
tgcgacaaga gcctgcacac cctgttcggc gacaagctgt gcaccgtggc caccctgcgc 2280
gagacctacg gcgagatggc cgactgctgc gccaagcagg agcccgagcg caacgagtgc 2340
ttcctgcagc acaaggacga caaccccaac ctgccccgcc tggtgcgccc cgaggtggac 2400
gtgatgtgca ccgccttcca cgacaacgag gagaccttcc tgaagaagta cctgtacgag 2460
atcgcccgcc gccaccccta cttctacgcc cccgagctgc tgttcttcgc caagcgctac 2520
aaggccgcct tcaccgagtg ctgccaggcc gccgacaagg ccgcctgcct gctgcccaag 2580
ctggacgagc tgcgcgacga gggcaaggcc agcagcgcca agcagcgcct gaagtgcgcc 2640
agcctgcaga agttcggcga gcgcgccttc aaggcctggg ccgtggcccg cctgagccag 2700
cgcttcccca aggccgagtt cgccgaggtg agcaagctgg tgaccgacct gaccaaggtg 2760
cacaccgagt gctgccacgg cgacctgctg gagtgcgccg acgaccgcgc cgacctggcc 2820
aagtacatct gcgagaacca ggacagcatc agcagcaagc tgaaggagtg ctgcgagaag 2880
cccctgctgg agaagagcca ctgcatcgcc gaggtggaga acgacgagat gcccgccgac 2940
ctgcccagcc tggccgccga cttcgtggag agcaaggacg tgtgcaagaa ctacgccgag 3000
gccaaggacg tgttcctggg catgttcctg tacgagtacg cccgccgcca ccccgactac 3060
agcgtggtgc tgctgctgcg cctggccaag acctacgaga ccaccctgga gaagtgctgc 3120
gccgccgccg acccccacga gtgctacgcc aaggtgttcgacgagttcaa gcccctggtg 3180
gaggagcccc agaacctgat caagcagaac tgcgagctgt tcgagcagct gggcgagtac 3240
aagttccaga acgccctgct ggtgcgctac accaagaagg tgccccaggt gagcaccccc 3300
accctggtgg aggtgagccg caacctgggc aaggtgggca gcaagtgctg caagcacccc 3360
gaggccaagc gcatgccctg cgccgaggac tacctgagcg tggtgctgaa ccagctgtgc 3420
gtgctgcacg agaagacccc cgtgagcgac cgcgtgacca agtgctgcac cgagagcctg 3480
gtgaaccgcc gcccctgctt cagcgccctg gaggtggacg agacctacgt gcccaaggag 3540
ttcaacgccg agaccttcac cttccacgcc gacatctgca ccctgagcga gaaggagcgc 3600
cagatcaaga agcagaccgc cctggtggag ctggtgaagc acaagcccaa ggccaccaag 3660
gagcagctga aggccgtgat ggacgacttc gccgccttcg tggagaagtg ctgcaaggcc 3720
gacgacaagg agacctgctt cgccgaggag ggcaagaagc tggtggccgc cagccaggcc 3780
gccctgggcc tgggaattct gcagatatcc agcacagtgg cggccgctcg agtc 3834
<210> 4
<211> 97
<212> DNA
<213> S-F0
<400> 4
acccaagctt gccaccatgg agacagacac actcctgcta tgggtactgc tgctctgggt 60
tccaggttcc accggttcct cacagtgcgt caatctg 97
<210> 5
<211> 57
<212> DNA
<213> S1-R1
<400> 5
cctcgctctt gtgggcgtca cctcctggcg cgcccgcggc tcttctggga gagtttg 57
<210> 6
<211> 54
<212> DNA
<213> HSA-F1
<400> 6
ccagaagagc cgcgggcgcg ccaggaggtg acgcccacaa gagcgaggtg gccc 54
<210> 7
<211> 36
<212> DNA
<213> HSA-R2
<400> 7
atctgcagaa ttcccaggcc cagggcggcc tggctg 36
<210> 8
<211> 57
<212> DNA
<213> S1-R1'
<400> 8
cctcgctctt gtgggcgtca cctcctggcg cgcctctggc tcttctggga gagtttg 57
<210> 9
<211> 54
<212> DNA
<213> HSA-F1'
<400> 9
ccagaagagc cagaggcgcg ccaggaggtg acgcccacaa gagcgaggtg gccc 54
<210> 10
<211> 36
<212> DNA
<213> HSA-R2'
<400> 10
atctgcagaa ttcccaggcc cagggcggcc tggctg 36
SEQUENCE LISTING
<110> Siger (Suzhou) Biotech Co., ltd
<120> a novel coronavirus antigen, and preparation method and application thereof
<130> 1234
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 674
<212> PRT
<213> amino acid sequence of Spike protein S1 Domain
<400> 1
Ser Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala
1 5 10 15
Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe
20 25 30
Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe
35 40 45
Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly
50 55 60
Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr
65 70 75 80
Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly
85 90 95
Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala
100 105 110
Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro
115 120 125
Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser
130 135 140
Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val
145 150 155 160
Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys
165 170 175
Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile
180 185 190
Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly
195 200 205
Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile
210 215 220
Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro
225 230 235 240
Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val
245 250 255
Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly
260 265 270
Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr
275 280 285
Lys Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr
290 295 300
Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn
305 310 315 320
Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe
325 330 335
Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala
340 345 350
Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys
355 360 365
Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val
370 375 380
Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala
385 390 395 400
Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp
405 410 415
Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser
420 425 430
Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser
435 440 445
Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala
450 455 460
Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro
465 470 475 480
Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro
485 490 495
Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr
500 505 510
Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val
515 520 525
Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser
530 535 540
Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp
545 550 555 560
Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile
565 570 575
Thr Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn
580 585 590
Thr Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu
595 600 605
Val Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val
610 615 620
Tyr Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile
625 630 635 640
Gly Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly
645 650 655
Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg
660 665 670
Ala Ala
<210> 2
<211> 1278
<212> PRT
<213> amino acid sequence of fusion protein
<400> 2
Ser Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala
1 5 10 15
Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe
20 25 30
Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe
35 40 45
Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly
50 55 60
Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr
65 70 75 80
Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly
85 90 95
Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala
100 105 110
Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro
115 120 125
Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser
130 135 140
Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val
145 150 155 160
Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys
165 170 175
Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile
180 185 190
Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly
195 200 205
Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile
210 215 220
Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro
225 230 235 240
Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val
245 250 255
Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly
260 265 270
Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr
275 280 285
Lys Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr
290 295 300
Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn
305 310 315 320
Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe
325 330 335
Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala
340 345 350
Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys
355 360 365
Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val
370 375 380
Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala
385 390 395 400
Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp
405 410 415
Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser
420 425 430
Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser
435 440 445
Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala
450 455 460
Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro
465 470 475 480
Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro
485 490 495
Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr
500 505 510
Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val
515 520 525
Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser
530 535 540
Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp
545 550 555 560
Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile
565 570 575
Thr Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn
580 585 590
Thr Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu
595 600 605
Val Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val
610 615 620
Tyr Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile
625 630 635 640
Gly Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly
645 650 655
Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg
660 665 670
Ala Ala Gly Ala Pro Gly Gly Asp Ala His Lys Ser Glu Val Ala His
675 680 685
Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile
690 695 700
Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys
705 710 715 720
Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu
725 730 735
Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys
740 745 750
Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp
755 760 765
Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His
770 775 780
Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp
785 790 795 800
Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys
805 810 815
Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu
820 825 830
Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys
835 840 845
Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu
850 855 860
Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala
865 870 875 880
Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala
885 890 895
Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys
900 905 910
Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp
915 920 925
Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys
930 935 940
Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys
945 950 955 960
Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu
965 970 975
Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys
980 985 990
Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met
995 1000 1005
Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val
1010 1015 1020
Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys
1025 1030 1035
Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe
1040 1045 1050
Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys
1055 1060 1065
Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln
1070 1075 1080
Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser
1085 1090 1095
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly
1100 1105 1110
Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala
1115 1120 1125
Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
1130 1135 1140
Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu
1145 1150 1155
Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp
1160 1165 1170
Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe
1175 1180 1185
His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys
1190 1195 1200
Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala
1205 1210 1215
Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe
1220 1225 1230
Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala
1235 1240 1245
Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly
1250 1255 1260
Leu Gly Ile Leu Gln Ile Ser Ser Thr Val Ala Ala Ala Arg Val
1265 1270 1275
<210> 3
<211> 3834
<212> DNA
<213> nucleotide sequence encoding fusion protein
<400> 3
tcctcacagt gcgtcaatct gacaactcgg actcagctgc cacctgctta tactaatagc 60
ttcaccagag gcgtgtacta tcctgacaag gtgtttagaa gctccgtgct gcactctaca 120
caggatctgt ttctgccatt ctttagcaac gtgacctggt tccacgccat ccacgtgagc 180
ggcaccaatg gcacaaagcg gttcgacaat cccgtgctgc cttttaacga tggcgtgtac 240
ttcgcctcta ccgagaagag caacatcatc agaggatgga tctttggcac cacactggac 300
tccaagacac agtctctgct gatcgtgaac aatgccacca acgtggtcat caaggtgtgc 360
gagttccagt tttgtaatga tcccttcctg ggcgtgtact atcacaagaa caataagagc 420
tggatggagt ccgagtttag agtgtattct agcgccaaca actgcacatt tgagtacgtg 480
agccagcctt tcctgatgga cctggagggc aagcagggca atttcaagaa cctgagggag 540
ttcgtgttta agaatatcga cggctacttc aaaatctact ctaagcacac ccccatcaac 600
ctggtgcgcg acctgcctca gggcttcagc gccctggagc ccctggtgga tctgcctatc 660
ggcatcaaca tcacccggtt tcagacactg ctggccctgc acagaagcta cctgacaccc 720
ggcgactcct ctagcggatg gaccgccggc gctgccgcct actatgtggg ctacctccag 780
ccccggacct tcctgctgaa gtacaacgag aatggcacca tcacagacgc agtggattgc 840
gccctggacc ccctgagcga gacaaagtgt acactgaagt cctttaccgt ggagaagggc 900
atctatcaga catccaattt cagggtgcag ccaaccgagt ctatcgtgcg ctttcctaat 960
atcacaaacc tgtgcccatt tggcgaggtg ttcaacgcaa cccgcttcgc cagcgtgtac 1020
gcctggaata ggaagcggat cagcaactgc gtggccgact atagcgtgct gtacaactcc 1080
gcctctttca gcacctttaa gtgctatggc gtgtccccca caaagctgaa tgacctgtgc 1140
tttaccaacg tctacgccga ttctttcgtg atcaggggcg acgaggtgcg ccagatcgcc 1200
cccggccaga caggcaagat cgcagactac aattataagc tgccagacga tttcaccggc 1260
tgcgtgatcg cctggaacag caacaatctg gattccaaag tgggcggcaa ctacaattat 1320
ctgtaccggc tgtttagaaa gagcaatctg aagcccttcg agagggacat ctctacagaa 1380
atctaccagg ccggcagcac cccttgcaat ggcgtggagg gctttaactg ttatttccca 1440
ctccagtcct acggcttcca gcccacaaac ggcgtgggct atcagcctta ccgcgtggtg 1500
gtgctgagct ttgagctgct gcacgcccca gcaacagtgt gcggccccaa gaagtccacc 1560
aatctggtga agaacaagtg cgtgaacttc aacttcaacg gcctgaccgg cacaggcgtg 1620
ctgaccgagt ccaacaagaa gttcctgcca tttcagcagt tcggcaggga catcgcagat 1680
accacagacg ccgtgcgcga cccacagacc ctggagatcc tggacatcac accctgctct 1740
ttcggcggcg tgagcgtgat cacacccggc accaatacaa gcaaccaggt ggccgtgctg 1800
tatcaggacg tgaattgtac cgaggtgccc gtggctatcc acgccgatca gctgacccca 1860
acatggcggg tgtacagcac cggctccaac gtcttccaga caagagccgg atgcctgatc 1920
ggagcagagc acgtgaacaa ttcctatgag tgcgacatcc caatcggcgc cggcatctgt 1980
gcctcttacc agacccagac aaactctccc agaagagccg cgggcgcgcc aggaggtgac 2040
gcccacaaga gcgaggtggc ccaccgcttc aaggacctgg gcgaggagaa cttcaaggcc 2100
ctggtgctga tcgccttcgc ccagtacctg cagcagtgcc ccttcgagga ccacgtgaag 2160
ctggtgaacg aggtgaccga gttcgccaag acctgcgtgg ccgacgagag cgccgagaac 2220
tgcgacaaga gcctgcacac cctgttcggc gacaagctgt gcaccgtggc caccctgcgc 2280
gagacctacg gcgagatggc cgactgctgc gccaagcagg agcccgagcg caacgagtgc 2340
ttcctgcagc acaaggacga caaccccaac ctgccccgcc tggtgcgccc cgaggtggac 2400
gtgatgtgca ccgccttcca cgacaacgag gagaccttcc tgaagaagta cctgtacgag 2460
atcgcccgcc gccaccccta cttctacgcc cccgagctgc tgttcttcgc caagcgctac 2520
aaggccgcct tcaccgagtg ctgccaggcc gccgacaagg ccgcctgcct gctgcccaag 2580
ctggacgagc tgcgcgacga gggcaaggcc agcagcgcca agcagcgcct gaagtgcgcc 2640
agcctgcaga agttcggcga gcgcgccttc aaggcctggg ccgtggcccg cctgagccag 2700
cgcttcccca aggccgagtt cgccgaggtg agcaagctgg tgaccgacct gaccaaggtg 2760
cacaccgagt gctgccacgg cgacctgctg gagtgcgccg acgaccgcgc cgacctggcc 2820
aagtacatct gcgagaacca ggacagcatc agcagcaagc tgaaggagtg ctgcgagaag 2880
cccctgctgg agaagagcca ctgcatcgcc gaggtggaga acgacgagat gcccgccgac 2940
ctgcccagcc tggccgccga cttcgtggag agcaaggacg tgtgcaagaa ctacgccgag 3000
gccaaggacg tgttcctggg catgttcctg tacgagtacg cccgccgcca ccccgactac 3060
agcgtggtgc tgctgctgcg cctggccaag acctacgaga ccaccctgga gaagtgctgc 3120
gccgccgccg acccccacga gtgctacgcc aaggtgttcg acgagttcaa gcccctggtg 3180
gaggagcccc agaacctgat caagcagaac tgcgagctgt tcgagcagct gggcgagtac 3240
aagttccaga acgccctgct ggtgcgctac accaagaagg tgccccaggt gagcaccccc 3300
accctggtgg aggtgagccg caacctgggc aaggtgggca gcaagtgctg caagcacccc 3360
gaggccaagc gcatgccctg cgccgaggac tacctgagcg tggtgctgaa ccagctgtgc 3420
gtgctgcacg agaagacccc cgtgagcgac cgcgtgacca agtgctgcac cgagagcctg 3480
gtgaaccgcc gcccctgctt cagcgccctg gaggtggacg agacctacgt gcccaaggag 3540
ttcaacgccg agaccttcac cttccacgcc gacatctgca ccctgagcga gaaggagcgc 3600
cagatcaaga agcagaccgc cctggtggag ctggtgaagc acaagcccaa ggccaccaag 3660
gagcagctga aggccgtgat ggacgacttc gccgccttcg tggagaagtg ctgcaaggcc 3720
gacgacaagg agacctgctt cgccgaggag ggcaagaagc tggtggccgc cagccaggcc 3780
gccctgggcc tgggaattct gcagatatcc agcacagtgg cggccgctcg agtc 3834
<210> 4
<211> 97
<212> DNA
<213> S-F0
<400> 4
acccaagctt gccaccatgg agacagacac actcctgcta tgggtactgc tgctctgggt 60
tccaggttcc accggttcct cacagtgcgt caatctg 97
<210> 5
<211> 57
<212> DNA
<213> S1-R1
<400> 5
cctcgctctt gtgggcgtca cctcctggcg cgcccgcggc tcttctggga gagtttg 57
<210> 6
<211> 54
<212> DNA
<213> HSA-F1
<400> 6
ccagaagagc cgcgggcgcg ccaggaggtg acgcccacaa gagcgaggtg gccc 54
<210> 7
<211> 36
<212> DNA
<213> HSA-R2
<400> 7
atctgcagaa ttcccaggcc cagggcggcc tggctg 36
<210> 8
<211> 57
<212> DNA
<213> S1-R1'
<400> 8
cctcgctctt gtgggcgtca cctcctggcg cgcctctggc tcttctggga gagtttg 57
<210> 9
<211> 54
<212> DNA
<213> HSA-F1'
<400> 9
ccagaagagc cagaggcgcg ccaggaggtg acgcccacaa gagcgaggtg gccc 54
<210> 10
<211> 36
<212> DNA
<213> HSA-R2'
<400> 10
atctgcagaa ttcccaggcc cagggcggcc tggctg 36

Claims (6)

1. A fusion protein comprising a Spike protein S1 domain portion and an HSA portion, the amino acid sequence of the Spike protein S1 domain portion being SEQ ID No ： 1, the amino acid sequence of the fusion protein is SEQ ID No ： 2；
Preparation of fusion protein S1-HSA:
the synthetic primers were as follows:
S-F0:ACCCAAGCTTgccaccATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGTTCCTCACAGTGCGTCAATCTG；
S1-R1: CCTCGCTCTTGTGGGCGTCACCTCCTGGCGCGCCCGCGGCTCTTCTGGGAGAGTTTG；
HSA-F1: CCAGAAGAGCCGCGGGCGCGCCAGGAGGTGACGCCCACAAGAGCGAGGTGGCCC；
HSA-R2: ATCTGCAGAATTCCCAGGCCCAGGGCGGCCTGGCTG；
the Spike gene is used as a template, S-F0/S1-R1 is used as a primer, S1 gene fragments are amplified, and amplification conditions are as follows: 94 ℃ for 5min; amplification system: 10Xbuffer, 5ul;2.5mM dNTP,2ul;10uM primer1,1ul;10uM primer2,1ul; template, 2ul; taq,0.5ul; ddH 2 O，38.5ul；
HSA gene is used as a template, HSA-F1/HSA-R2 is used as a primer, an HSA fragment is amplified, and amplification conditions are as follows: 94 ℃ for 5min;72 ℃ for 5min;
amplification system: 10Xbuffer, 5ul;2.5mM dNTP,2ul;10uM primer1,1ul;10uM primer2,1ul; template, 2ul; taq,0.5ul; ddH 2 O，38.5ul；
And then the amplified S1 fragment and the amplified HSA fragment are used as templates, S-F0 and HSA-R2 are used as primers, the S1-HSA fusion fragment is amplified, and the amplification conditions are as follows: 94 ℃ for 5min;72 ℃ for 5min;
amplification system: 10Xbuffer, 5ul;2.5mM dNTP,2ul;10uM primer1,1ul;10uM primer2,1ul; template, 2ul; taq,0.5ul; ddH 2 O，38.5ul；
The PCDNA3.1 plasmid vector was digested with HindIII/EcoRI and the large fragment was recovered using the gel recovery kit; the S1-HSA PCR product was digested with HindIII/EcoRI, the digestion system: buffer 2X: 2ul, ecoRI:0.5ul, hindIII:0.5ul, template PCR product 2ul, water: 15ul, reacting for 30 minutes at 37 ℃, and then recycling by using a glue recycling kit; connecting the vector and the fragment recovered by enzyme digestion by using T4 DNase for 30 minutes, transforming TOP10 escherichia coli competent of the connected products, culturing overnight at 37 ℃, and picking up clone for sequencing and identification the next day; for the correct sequencing clones, the recombinant plasmid in E.coli TOP10 was extracted with endotoxin-free plasmid extraction kit, designated: pCDNA3.1-S1-HAS;
to construct the pCDNA3.1-S1 (wt) -HSA plasmid, the synthetic primers were designed as follows:
S-F0:ACCCAAGCTTgccaccATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGTTCCTCACAGTGCGTCAATCTG；
S1-R1’：
CCTCGCTCTTGTGGGCGTCACCTCCTGGCGCGCCtctGGCTCTTCTGGGAGAGTTTG；
HSA-F1’：
CCAGAAGAGCCagaGGCGCGCCAGGAGGTGACGCCCACAAGAGCGAGGTGGCCC；
HSA-R2’：ATCTGCAGAATTCCCAGGCCCAGGGCGGCCTGGCTG；
s1 gene fragment is amplified by taking Spike gene as a template and S-F0/S1-R' as a primer, and the amplification conditions are as follows: 94 ℃ for 5min; amplification system: 10Xbuffer, 5ul;2.5mM dNTP,2ul;10uM primer1,1ul;10uM primer2,1ul; template, 2ul; taq,0.5ul; ddH 2 O,38.5ul; HSA gene is used as a template, HSA-F1 '/HSA-R2' is used as a primer, an HSA fragment is amplified, and amplification conditions are as follows: 94 ℃ for 5min; amplification system: 10Xbuffer, 5ul;2.5mM dNTP,2ul;10uM primer1,1ul;10uM primer2,1ul; template, 2ul; taq,0.5ul; ddH 2 O,38.5ul; then S1 fragment and HSA fragment after amplification are used as templates, S-F0 and HSA-R2' are used as primers, and S1-HSA fusion is amplifiedFragments; cutting PCDNA3.1 plasmid vector with HindIII/EcoRI, recovering large fragment by gel, cutting S1-HSA PCR product with HindIII/EcoRI, recovering gel, connecting the carrier and fragment by T4 DNase for 30 min, transforming TOP10 E.coli competent with the connected product, culturing overnight at 37 ℃, picking clone for the next day, sequencing and identifying; for the correct sequencing clones, the recombinant plasmid in E.coli TOP10 was extracted with endotoxin-free plasmid extraction kit, designated: pCDNA3.1-S1 (wt) -HAS;
293F cells were cultured until the cell density reached 2X 10 6 –3×10 6 Cells/ml were transfected with recombinant plasmid, cultured for 7 days after transfection, the supernatant was collected, centrifuged at high speed, and the fusion protein was purified by HSA affinity chromatography.
2. A nucleotide encoding the fusion protein of claim 1, wherein: the nucleotide sequence is SEQ ID No:3.
3. a recombinant vector, characterized in that: comprising the nucleotide of claim 2.
4. An expression cassette, characterized in that: comprising the nucleotide of claim 2.
5. A microorganism or host cell comprising the nucleotide of claim 2.
6. A pharmaceutical composition characterized by: comprising the fusion protein of claim 1.
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