CN113621076A - Fusion protein for Delta of new coronavirus variant strain, nasal spray type vaccine, and preparation method and application thereof - Google Patents

Fusion protein for Delta of new coronavirus variant strain, nasal spray type vaccine, and preparation method and application thereof Download PDF

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CN113621076A
CN113621076A CN202111011970.8A CN202111011970A CN113621076A CN 113621076 A CN113621076 A CN 113621076A CN 202111011970 A CN202111011970 A CN 202111011970A CN 113621076 A CN113621076 A CN 113621076A
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protein
vaccine
fusion protein
delta
nasal spray
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左建宏
谢卓熠
黄佳璐
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University of South China
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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • 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
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • 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

Abstract

The invention provides a fusion protein for a Delta variant strain of a new coronavirus, a nasal spray type vaccine, and a preparation method and application thereof, and belongs to the technical field of virus vaccines. A fusion protein aiming at a novel coronavirus variant strain Delta is formed by fusing a spike protein S, a transmembrane protein M and a nucleocapsid protein N; the coding genes of the S protein, the M protein and the N protein are shown as SEQ ID NO: 1 to 3. The nasal spray type vaccine prepared by mixing the fusion protein and the adjuvant has good immunogenicity, safety and biological activity, and can induce an organism to generate an effective antibody, so that the growth of a novel coronavirus variant Delta is inhibited, and the Delta can be effectively prevented from being infected; the nasal spray type vaccine is adopted, injection is not needed, the operation is simple and convenient, adverse reaction after the injection type vaccine is used is avoided, the preparation method of the vaccine is simple, the purification is easy, the safety is high, and the vaccine can be quickly applied to clinical tests.

Description

Fusion protein for Delta of new coronavirus variant strain, nasal spray type vaccine, and preparation method and application thereof
Technical Field
The invention belongs to the technical field of virus vaccines, and particularly relates to a fusion protein for a novel coronavirus variant strain Delta, a nasal spray type vaccine, and a preparation method and application thereof.
Background
SARS-CoV-2 (or 2019-nCoV) is a pathogenic virus of new coronary pneumonia. The Delta virus strain is a new coronavirus variant strain and has the characteristics of obviously enhanced transmission capability, shortened incubation period and generation interval and increased disease severity. At present, no special treatment medicine exists for the new coronary pneumonia caused by Delta variant infection. In the face of the difficulty in finding a cure, the development of vaccines has become an urgent task.
Existing vaccine preparations include traditional vaccines (attenuated live vaccines, inactivated vaccines), subunit vaccines, viral vector vaccines, nucleic acid vaccines, and the like. Among them, the recombinant subunit vaccine is a vaccine that induces an organism to produce nucleic acid-free antibodies using a certain surface structural component of a microorganism (antigen). These structures may include certain polysaccharides, proteins or epitopes, but they do not include the entire pathogen. On the basis of subunit vaccines, nano-particles are loaded to form nano-vaccines, which is also a popular way for vaccine design.
The Delta virus has extremely strong infectivity and high pathogenicity. Vaccines with a complete viral structure do not guarantee their safety and may cause ineffective antibody production in vaccinated subjects. The generation of ineffective antibodies often has adverse effects such as failure to respond to viral infection and even enhancement of dependent infection and respiratory disease. Single protein vaccines may not be well immunogenic or face the risk of failure in the face of infection with other mutants of the same type.
Disclosure of Invention
In view of the above, the present invention aims to provide a fusion protein for Delta of a novel coronavirus variant strain, a nasal spray vaccine, and a preparation method and application thereof, which have ideal immunogenicity, generate effective antibodies, and can prevent the Delta virus strain.
The invention provides a fusion protein for a new coronavirus variant strain Delta, which is formed by fusing an S protein, an M protein and an N protein;
the nucleotide sequence of the coding gene of the S protein is shown as SEQ ID NO: 1 is shown in the specification;
the nucleic acid sequence of the coding gene of the M protein is shown as SEQ ID NO: 2 is shown in the specification;
the nucleotide sequence of the coding gene of the N protein is shown as SEQ ID NO: 3, respectively.
Preferably, the fusion protein is an S-M-N fusion protein.
Preferably, a flexible linker is further included between every two proteins in the fusion protein;
the amino acid sequence of the flexible linker is (GGGGS) n, wherein n is an integer and is more than or equal to 1 and less than or equal to 3.
The invention provides a nasal spray type vaccine aiming at a novel coronavirus variant strain Delta, which comprises a fusion protein and an adjuvant.
Preferably, the concentration of the fusion protein is 0.1-0.2 g/L;
preferably, the adjuvant is PLGA.
Preferably, the final concentration of the PLGA in the vaccine is 1-2 mg/mL.
The invention provides application of the fusion protein in preparation of a vaccine aiming at a novel coronavirus variant strain Delta.
Preferably, the vaccine is a nasal spray vaccine.
The fusion protein for the Delta of the novel coronavirus variant strain is formed by fusing S protein, M protein and N protein; the nucleotide sequence of the S protein is shown as SEQ ID NO: 1 is shown in the specification; the nucleic acid sequence of the coding gene of the M protein is shown as SEQ ID NO: 2 is shown in the specification; the nucleotide sequence of the coding gene of the N protein is shown as SEQ ID NO: 3, respectively. Wherein, the spike protein S is a key protein for the infection of the new coronavirus combined with the human body, and the mutation site is the most and the most key; transmembrane protein M, nucleocapsid protein N are also the major structures that make up Delta. The fusion protein constructed by the invention has good immunogenicity and biological activity, and can induce organisms to generate effective antibodies, thereby inhibiting the growth of novel coronavirus variant (Delta) and effectively preventing Delta infection. Meanwhile, the fusion protein has higher safety compared with a whole virus candidate vaccine strain.
The invention provides a nasal spray type vaccine aiming at a novel coronavirus variant strain Delta, which comprises a fusion protein and an adjuvant. The components of the nasal spray type vaccine are coordinated and supplemented with each other, so that the nasal spray type vaccine has good immunogenicity, safety and biological activity, and can induce an organism to generate effective antibodies, thereby inhibiting the growth of novel coronavirus variant (Delta) and effectively preventing Delta infection. Meanwhile, the nasal spray type vaccine is adopted, injection is not needed, the operation is simple and convenient, and adverse reactions of pain and red swelling of injection parts after the injection type vaccine is used are avoided. The vaccine provided by the invention is simple in preparation method, easy to purify and high in safety, and the vaccine can be quickly applied to clinical tests.
Drawings
FIG. 1 is a diagram of a nasal spray vaccine cartridge for holding a nasal spray vaccine according to the present invention;
FIG. 2 is a scanning electron microscope image of a novel coronavirus S-M-N vaccine of the present invention;
FIG. 3 is a graph of the in vitro release of S-M-N provided by a vaccine embodying the present invention;
FIG. 4 is a bar graph of antibodies against S, M, N protein produced by S-M-N vaccines according to the present invention;
FIG. 5 is a histogram of the cytokine IL-2 produced in vivo by S-M-N vaccines of the present invention after immunization of mice.
Detailed Description
The invention provides a fusion protein for a new coronavirus variant strain Delta, which is formed by fusing an S protein, an M protein and an N protein; the nucleotide sequence of the coding gene of the S protein is shown as SEQ ID NO: 1
(tataattataaattaccagatgattttacaggctgcgttatagcttggaattctaacaatcttgattctaaggttggtggtaattataattaccggtatagattgtttaggaagtctaatctcaaaccttttgagagagatatttcaactgaaatctatcaggccggtagcaaaccttgtaatggtgttgaaggttttaattgttactttcctttacaatcatatggtttccaacccactaatggtgttggttaccaaccatacagagtagtagtactttcttttgaacttctacatgcaccagcaactgtttgtggacctaaaaagtctactaatttggttaaaaacaaatgtgtcaatttcaacttcaatggtttaacaggcacaggtgttcttactgagtctaacaaaaagtttctgcctttccaacaatttggcagagacattgctgacactactgatgctgtccgtgatccacagacacttgagattcttgacattacaccatgttcttttggtggtgtcagtgttataacaccaggaacaaatacttctaaccaggttgctgttctttatcagggtgttaactgcacagaagtccctgttgctattcatgcagatcaacttactcctacttggcgtgtttattctacaggttctaatgtttttcaaacacgtaatcttttgttgcaatatggcagtttttgtacacaattaaaccgtgctttaactggaatagctgttgaacaagacaaaaacacccaagaagtttttgcacaagtcaaacaaatttacaaaacaccaccaattaaagattttggtggttttaatttttcacaaatattaccagatccatcaaaaccaagcaagaggtcatttattgaagatctacttttcaacaaagtgacacttgcagatgctggcttcatcaaacaatatggtgattgccttggtgatattgctgctagagacctcatttgtgcacaaaagtttaacggccttactgttttgccacctttgctcacagatgaaatgattgctcaatacacttctgcactgttagcgggtacaatcacttctggttggacctttggtgcaggtgctgcattacaaataccatttgctatgcaaatggcttataggtttaatggtattggagttacacagaatgttctctatgagaaccaaaaattgattgccaaccaatttaatagtgctattggcaaaattcaagactcactttcttccacagcaagtgcacttggaaaacttcaaaatgtggtcaaccaaaatgcacaagctttaaacacgcttgttaaacaacttagctccaattttggtgcaatttcaagtgttttaaatgatatcctttcacgtcttgacaaagttgaggctgaagtgcaaattgataggttgatcacaggcagacttcaaagtttgcagacatatgtgactcaacaattaattagagctgcagaaatcagagcttctgctaatcttgctgctactaaaatgtcagagtgtgtacttggacaatcaaaaagagttgatttttgtggaaagggctatcatcttatgtccttccctcagtcagcacctcatggtgtagtcttcttgcatgtgacttatgtccctgcacaagaaaagaacttcacaactgctcctgccatttgtcatgatggaaaagcacactttcctcgtgaaggtgtctttgtttcaaatggcacacactggtttgtaacacaaaggaatttttatgaaccacaaatcattactacagacaacacatttgtgtctggtaactgtgatgttgtaataggaattgtcaacaacacagtttatgatcctttgcaacctgaattagactcattcaaggaggagttagataaatattttaagaatcatacatcaccagatg); the nucleic acid sequence of the coding gene of the M protein is shown as SEQ ID NO: 2 (atggcagattccaacggtactattaccgttgaagagcttaaaaagctccttgaacaatggaacctagtaataggtttcctattccttacatggatttgtcttctacaatttgcctatgccaacaggaataggtttttgtatataattaagttaattttcctctggctgttatggccagtaactttagcttgttttgtgcttgctgctgtttacagaataaattggatcaccggtggaatttctaccgcaatggcttgtcttgtaggcttgatgtggctcagctacttcattgcttctttcagactgtttgcgcgtacgcgttccatgtggtcattcaatccagaaactaacattcttctcaacgtgccactccatggcactattctgaccagaccgcttctagaaagtgaactcgtaatcggagctgtgatccttcgtggacatcttcgtattgctggacaccatctaggacgctgtgacatcaaggacctgcctaaagaaatcactgttgctacatcacgaacgctttcttattacaaattgggagcttcgcagcgtgtagcaggtgactcaggttttgctgcatacagtcgctacaggattggcaactataaattaaacacagaccattccagtagcagtgacaatattgctttgcttgtacagtaa); the nucleotide sequence of the coding gene of the N protein is shown as SEQ ID NO: 3 (atgtctgataatggaccccaaaatcagcgaaatgcaccccgcattacgtttggtggaccctcagattcaactggcagtaaccagaatggagaacgcagtggggcgcgatcaaaacaacgtcggccccaaggtttacccaataatactgcgtcttggttcaccgctctcactcaacatggcaaggaaggccttaaattccctcgaggacaaggcgttccaattaacaccaatagcagtccagatgaccaaattggctactaccgaagagctaccagacgaattcgtggtggtgacggtaaaatgaaagatctcagtccaagatggtatttctactacctaggaactgggccagaagctggacttccctatggtgctaacaaagacggcatcatatgggttgcaactgagggagccttgaatacaccaaaagatcacattggcacccgcaatcctgctaacaatgctgcaatcgtgctacaacttcctcaaggaacaacattgccaaaaggcttctacgcagaagggagcagaggcggcagtcaagcctcttctcgttcctcatcacgtagtcgcaacagttcaagaaattcaactccaggcagcagtatgggaacttctcctgctagaatggctggcaatggctgtgatgctgctcttgctttgctgctgcttgacagattgaaccagcttgagagcaaaatgtctggtaaaggccaacaacaacaaggccaaactgtcactaagaaatctgctgctgaggcttctaagaagcctcggcaaaaacgtactgccactaaagcatacaatgtaacacaagctttcggcagacgtggtccagaacaaacccaaggaaattttggggaccaggaactaatcagacaaggaactgattacaaacattggccgcaaattgcacaatttgcccccagcgcttcagcgttcttcggaatgtcgcgcattggcatggaagtcacaccttcgggaacgtggttgacctacacaggtgccatcaaattggatgacaaagatccaaatttcaaagatcaagtcattttgctgaataagcatattgacgcatacaaa).
In the present invention, the connection order of the three proteins in the fusion protein is not particularly limited, and any known connection manner of the three proteins may be adopted. To illustrate the performance of the fusion protein, in the examples of the present invention, the fusion protein was prepared in the order of S-M-N. In the present invention, in order to ensure the spatial folding of each protein in the fusion protein and to improve the biological activity of each protein, it is preferable that a flexible linker is further included between every two proteins in the fusion protein. The amino acid sequence of the flexible linker is (GGGGS) n, wherein n is an integer and is more than or equal to 1 and less than or equal to 4. In the embodiment of the present invention, the amino acid sequence of the flexible linker is preferably GGGGSGGGGSGGGGS (SEQ ID NO: 4).
In the present invention, the method for constructing the fusion protein preferably comprises the following steps:
1) obtaining a fusion gene segment with a restriction enzyme site by using a nucleotide sequence of an artificially synthesized coding gene of the fusion protein;
2) carrying out enzyme digestion on the fusion gene segment with the enzyme digestion site in the step 1) and the gene expression vector respectively to obtain an amplified segment and a linear expression vector;
3) connecting the amplified fragment in the step 2) with a linear expression vector to obtain a recombinant vector;
4) and carrying out recombinant expression on the recombinant vector through a prokaryotic expression system, and purifying to obtain the recombinant expressed fusion protein.
The type of gene expression vector used in the present invention is not particularly limited, and any type of gene expression vector known in the art may be used. In the present embodiment, the gene expression vector is preferably pET-28 a. The enzyme for enzyme cleavage is preferably XholI. The XholI enzyme is not limited by the conditions of the present invention, and the enzyme digestion method known in the art can be used. And preferably purifying by using a gene purification kit after enzyme digestion. And obtaining the purified amplified fragment and a linear expression vector, and then connecting. The enzyme used for the ligation is preferably T4 ligase. The ligation conditions of the T4 ligase in the present invention are not limited to those in the art. The recombinant vector is preferably verified before recombinant expression. The verification method preferably adopts the primer to amplify and sequence the recombinant vector as a template, and the sequencing result is consistent with the sequence of the target gene, which indicates that the recombinant vector is successfully constructed.
The method for the recombinant expression of the prokaryotic expression system is not particularly limited, and the method for the recombinant expression of the prokaryotic expression system known in the field can be adopted. After recombinant expression, lysis is preferably performed using a lysis solution. The lysate comprises SDS Lysis buffer. The volume of the lysate to the cells is preferably 1: 1. The purification method of the present invention is not particularly limited, and those in the art can be usedKnown purification methods. In the present example, the purification is preferably carried out using a molecular sieve column. The purification uses a chromatographic column, and the chromatographic column is Ni2+A metal chelating chromatography column. The conditions for the purification by the column are not particularly limited, and a purification method known in the art may be used. The inclusion body of the Rostta (DE3) pET28a-S-M-N genetic engineering bacteria is obtained after purification.
The invention provides a nasal spray type vaccine aiming at a novel coronavirus variant strain Delta, which comprises a fusion protein and an adjuvant.
In the invention, the concentration of the fusion protein is preferably 0.1-0.2 g/L, and more preferably 0.15 g/L. The adjuvant is preferably PLGA. The final concentration of the PLGA in the vaccine is preferably 1-2 mg/mL, more preferably 1.2-1.8 mg/mL, and most preferably 1.5 mg/mL. The PLGA has the following advantages as an adjuvant: (I) PLGA possesses good biodegradability and biocompatibility, (ii) the adequate preparation and production methods described are applicable to a wide variety of drugs, such as hydrophilic or hydrophobic molecules or macromolecules, (iii) degradation of vaccine antigens can be reduced, (iv) sustained release potential, (v) surface properties can be altered, stealth can be provided and/or better interaction with biomaterials can be achieved.
In the present invention, the preparation method of the nasal spray vaccine preferably comprises the following steps:
mixing the recombinant expressed fusion protein with an adjuvant to obtain colostrum, mixing with PVA, washing with double distilled water, dissolving in Tris buffer solution, and removing the solvent to obtain the nasal spray type vaccine.
In the present invention, the adjuvant is preferably PLGA. The adjuvant is preferably mixed with the fusion protein in the form of a 50mg/ml solution of PLGA in methylene chloride. The mass ratio of the fusion protein to the PLGA is 1:12.5 in a U-shaped mode. The solvent for the fusion protein is preferably a PBS solution.
The invention provides application of the fusion protein in preparation of a vaccine aiming at a novel coronavirus variant strain Delta. The vaccine is preferably a nasal spray vaccine. The preparation method of the vaccine is the same as the above record, and is not repeated herein.
In the present invention, the nasal spray vaccine is used as follows: spraying the prepared nasal spray type vaccine into nasal cavity.
The fusion protein of a variant strain Delta of the novel coronavirus, the nasal spray vaccine, the preparation method and the application thereof provided by the invention are described in detail in the following with reference to the examples, but the fusion protein and the nasal spray vaccine cannot be understood as limiting the protection scope of the invention.
Example 1
Preparation method of S-M-N protein vaccine
1 find S, M, N amino acids of Delta virus in the Uniprot protein database, and link the 5 'sequence of S protein and the 3' sequence of M protein, and the 5 'sequence of M protein and the 3' sequence of N protein with flexible linker. The linker is composed of glycine G and serine S (GGGGS)3。
2, carrying out enzyme digestion on 5 'and 3' ends of the coding gene of the S-M-N protein by Xhol I, utilizing T4 ligase pET-28a plasmid to connect, transforming into competent cells to obtain an S-M-N gene expression plasmid, and obtaining a cell strain of the stably expressed recombinant S-M-N protein through monoclonal screening.
3, expanding and culturing the cell strain, and carrying out secretory expression and purification to obtain the purified recombinant Delta S-M-N protein.
3.1 inoculating the recombinant S-M-N protein cell line in 50. mu.g/ml kanamycin Lysis Broth (LB), shaking at 37 ℃ and 160rpm overnight, inoculating the genetically engineered bacteria colony in LB/kam culture solution according to 3% of inoculum size, shaking at 200-300 rpm to OD600When the concentration is 0.5-0.7, IPTG is added to make the final concentration 0.5mM, after 4-6 hours of induction, 2000-4000 g is centrifuged for 5-15 min at 2-6 ℃, supernatant is removed, SDS lysine Buffer with corresponding volume is used for cracking, the mixture is mixed evenly at room temperature, and the cracking is carried out at 4 ℃.
3.2, the lysate is placed in an ice bath for 5-20 times of 50-55W ultrasound, the ultrasound is carried out for 20-40S each time, the operation is stopped for 20-40S until the lysate is not viscous, the lysate is centrifuged for 10-20 min at 12000g and 4 ℃, and the supernatant is removed, so that the Rostta (DE3) pET28a-S-M-N genetic engineering bacteria inclusion body is obtained.
4 use of Ni2+Metal chelating chromatographic column, purifying protein, and concrete stripThe following parts:
1)0.05mol/LEDTA, 0.5mol/LNaCl solution 100 mL;
2) 50mL of 2mol/LNaCl solution;
3) 50mL of 1mol/LNaOH solution;
4)0.2mol/LNiSO450mL of the solution;
5) and (3) an equilibrium buffer: 50mmol/LTris-HCl, 500mmol/LNaCl, pH 7.0500 mL;
6)Ni2+Chelating Sepharose FastFlow 5～10mL；
7) 20-50 ml of recombinant S-M-N protein sample;
8) washing liquid: 50mmol/L imidazole, 50mmol/L Tris-HCl, 500mmol/L NaCl, pH 7.0;
9) eluent: 300mmol/L imidazole, 50mmol/L Tris-HCl, 500mmol/L NaCl, pH 7.0
10) 50ml of 20% ethanol solution.
5-10 mL of affinity chromatography agent; peristaltic pump flow rate was 15rpm (about 2 ml/min); 120s for collection.
5 preparation of S-M-N protein vaccine
Weighing 100mg of PLGA and dissolving in 2ml of dichloromethane; taking the S-M-N protein solution, adding 2ml PBS, wherein the S-M-N protein content is 8 mg; mixing the two solutions, stirring with a magnetic stirrer at 400rpm for 2min to form primary emulsion, adding PVA with the final concentration of 2%, 300bar and 2min, washing with double distilled water, dissolving in 10ml Tris buffer solution, stirring at room temperature for 3-5 h to fully volatilize the organic solvent, centrifuging at 12000rpm for 10min to collect particles, and washing with double distilled water to obtain PLGA vaccine nanoparticles (PLGA-S-M-N nanopowder).
Example 2
Encapsulation rate determination and characterization analysis observation of S-M-N vaccine
1 nanoparticle drug loading and encapsulation efficiency
Determination of S-M-N protein concentration by BCA protein quantification assay
10mg of PLGA-S-M-N nanopowder prepared as described above was weighed in 2ml of an aqueous solution of 0.05mol/L NaOH and 1% SDS, centrifuged, and the amount of protein contained in the supernatant was measured, and the drug loading (see formula I) and encapsulation efficiency (see formula II) were calculated as 8.2% and 63.2%, respectively, by subtracting free protein from total protein.
Drug Loading (LC) ═ total protein-free protein)/total nanoparticles × 100% formula I;
wherein the total protein is added with a fixed mass, and the free protein is used for measuring the mass of the S-M-N protein; the total nanoparticles are the mass of the nano-encapsulated protein;
encapsulation Efficiency (EE) ═ total protein-free protein/total protein x 100% formula II
Wherein, the total protein is the total protein added with fixed mass, and the free protein is the mass of the protein S-M-N to be measured.
2 scanning electron microscope of nano-particles
The surface morphology of the nanoparticles was observed by scanning electron microscopy SEM, which is shown in fig. 2, and it can be seen that the prepared nanoparticles are oval.
3S-M-N vaccine nanoparticle release
As shown in FIG. 3, the S-M-N vaccine nanoparticles prepared in this example had a rapid release within 24h in the first stage, and the release rate was 13.2%. In the second phase, after 24h, diffusion-driven S-M-N is released continuously through the rigid PLGA core. At 84h, the cumulative release rate of the S1-E vaccine (see formula III) reached a peak of 63.4% with complete release.
Release rate (%) — the supernatant protein concentration/total protein concentration was measured every 12h 100% formula III.
Example 3
S-M-N protein vaccine prepared in example 1 was used in mouse immune inhalation test
Female BALB/c mice 6-8 weeks old were divided into 2 groups (S-M-N-PLGA vaccine particles group, negative control group), and 8 mice per group were each inhaled through the nose. Each vaccine was dissolved in PBS at a volume of 200. mu.L. Blood was drawn from the orbit of the mouse and serum was isolated for antibody detection. Serum samples were stored at-20 ℃ until use.
The indirect ELISA method detects the Delta-specific IgG antibody level and the sum of antibody titer in the serum of the immunized mice. ELISA plates were coated with either S, M or N protein at 20. mu.g/ml overnight at 4 ℃. Washing with 0.05% PBST three times, blocking with 1% BSA-PBST blocking solution, and standing at room temperatureIncubate for 1 h. Washing with 0.05% PBST for 5 times, adding 200 μ l diluted serum (1: 1000 dilution), and incubating at 37 deg.C for 2 h; PBST was washed 5 times, added with HRP-goat anti-mouse IgG (1: 100 dilution), 200. mu.l/well and incubated for 1h at room temperature; PBST was washed 5 times, 200. mu.l of substrate solution was added, incubated at room temperature in the dark for 20 minutes to develop color, and then 50. mu.l of stop buffer was added to the mixture to stop the color development at OD450nmAnd (6) reading.
As shown in FIG. 4, the experimental group OD450nmHigher than the control group. The results show that after the S-M-N vaccine provided by the embodiment is used for immunizing mice, high-level Delta-specific serum IgG is induced in the mice, and the induction is obviously higher than that of a control group.
Example 4
Cytokine detection
The expression of immunocytokines was measured in the mouse splenocytes (mice treated in example 3) by ELISA, and the immunocytokines level in the negative control group was also measured.
The result shows that the spleen cells CD4 of the mouse experimental group+T cell, CD8+T cells are remarkably increased, IL-2 is also remarkably changed (see figure 5), and the T cells are remarkably increased compared with a control group. The results show that the mice generate strong cellular immune response under the stimulation of the S-M-N protein vaccine of the invention.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> university of southern China
<120> fusion protein aiming at Delta of new coronavirus variant strain, nasal spray type vaccine, preparation method and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1918
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tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 60
cttgattcta aggttggtgg taattataat taccggtata gattgtttag gaagtctaat 120
ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag caaaccttgt 180
aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 240
aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 300
ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 360
ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 420
cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 480
acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 540
ggaacaaata cttctaacca ggttgctgtt ctttatcagg gtgttaactg cacagaagtc 600
cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 660
aatgtttttc aaacacgtaa tcttttgttg caatatggca gtttttgtac acaattaaac 720
cgtgctttaa ctggaatagc tgttgaacaa gacaaaaaca cccaagaagt ttttgcacaa 780
gtcaaacaaa tttacaaaac accaccaatt aaagattttg gtggttttaa tttttcacaa 840
atattaccag atccatcaaa accaagcaag aggtcattta ttgaagatct acttttcaac 900
aaagtgacac ttgcagatgc tggcttcatc aaacaatatg gtgattgcct tggtgatatt 960
gctgctagag acctcatttg tgcacaaaag tttaacggcc ttactgtttt gccacctttg 1020
ctcacagatg aaatgattgc tcaatacact tctgcactgt tagcgggtac aatcacttct 1080
ggttggacct ttggtgcagg tgctgcatta caaataccat ttgctatgca aatggcttat 1140
aggtttaatg gtattggagt tacacagaat gttctctatg agaaccaaaa attgattgcc 1200
aaccaattta atagtgctat tggcaaaatt caagactcac tttcttccac agcaagtgca 1260
cttggaaaac ttcaaaatgt ggtcaaccaa aatgcacaag ctttaaacac gcttgttaaa 1320
caacttagct ccaattttgg tgcaatttca agtgttttaa atgatatcct ttcacgtctt 1380
gacaaagttg aggctgaagt gcaaattgat aggttgatca caggcagact tcaaagtttg 1440
cagacatatg tgactcaaca attaattaga gctgcagaaa tcagagcttc tgctaatctt 1500
gctgctacta aaatgtcaga gtgtgtactt ggacaatcaa aaagagttga tttttgtgga 1560
aagggctatc atcttatgtc cttccctcag tcagcacctc atggtgtagt cttcttgcat 1620
gtgacttatg tccctgcaca agaaaagaac ttcacaactg ctcctgccat ttgtcatgat 1680
ggaaaagcac actttcctcg tgaaggtgtc tttgtttcaa atggcacaca ctggtttgta 1740
acacaaagga atttttatga accacaaatc attactacag acaacacatt tgtgtctggt 1800
aactgtgatg ttgtaatagg aattgtcaac aacacagttt atgatccttt gcaacctgaa 1860
ttagactcat tcaaggagga gttagataaa tattttaaga atcatacatc accagatg 1918
<210> 2
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atggcagatt ccaacggtac tattaccgtt gaagagctta aaaagctcct tgaacaatgg 60
aacctagtaa taggtttcct attccttaca tggatttgtc ttctacaatt tgcctatgcc 120
aacaggaata ggtttttgta tataattaag ttaattttcc tctggctgtt atggccagta 180
actttagctt gttttgtgct tgctgctgtt tacagaataa attggatcac cggtggaatt 240
tctaccgcaa tggcttgtct tgtaggcttg atgtggctca gctacttcat tgcttctttc 300
agactgtttg cgcgtacgcg ttccatgtgg tcattcaatc cagaaactaa cattcttctc 360
aacgtgccac tccatggcac tattctgacc agaccgcttc tagaaagtga actcgtaatc 420
ggagctgtga tccttcgtgg acatcttcgt attgctggac accatctagg acgctgtgac 480
atcaaggacc tgcctaaaga aatcactgtt gctacatcac gaacgctttc ttattacaaa 540
ttgggagctt cgcagcgtgt agcaggtgac tcaggttttg ctgcatacag tcgctacagg 600
attggcaact ataaattaaa cacagaccat tccagtagca gtgacaatat tgctttgctt 660
gtacagtaa 669
<210> 3
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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atgtctgata atggacccca aaatcagcga aatgcacccc gcattacgtt tggtggaccc 60
tcagattcaa ctggcagtaa ccagaatgga gaacgcagtg gggcgcgatc aaaacaacgt 120
cggccccaag gtttacccaa taatactgcg tcttggttca ccgctctcac tcaacatggc 180
aaggaaggcc ttaaattccc tcgaggacaa ggcgttccaa ttaacaccaa tagcagtcca 240
gatgaccaaa ttggctacta ccgaagagct accagacgaa ttcgtggtgg tgacggtaaa 300
atgaaagatc tcagtccaag atggtatttc tactacctag gaactgggcc agaagctgga 360
cttccctatg gtgctaacaa agacggcatc atatgggttg caactgaggg agccttgaat 420
acaccaaaag atcacattgg cacccgcaat cctgctaaca atgctgcaat cgtgctacaa 480
cttcctcaag gaacaacatt gccaaaaggc ttctacgcag aagggagcag aggcggcagt 540
caagcctctt ctcgttcctc atcacgtagt cgcaacagtt caagaaattc aactccaggc 600
agcagtatgg gaacttctcc tgctagaatg gctggcaatg gctgtgatgc tgctcttgct 660
ttgctgctgc ttgacagatt gaaccagctt gagagcaaaa tgtctggtaa aggccaacaa 720
caacaaggcc aaactgtcac taagaaatct gctgctgagg cttctaagaa gcctcggcaa 780
aaacgtactg ccactaaagc atacaatgta acacaagctt tcggcagacg tggtccagaa 840
caaacccaag gaaattttgg ggaccaggaa ctaatcagac aaggaactga ttacaaacat 900
tggccgcaaa ttgcacaatt tgcccccagc gcttcagcgt tcttcggaat gtcgcgcatt 960
ggcatggaag tcacaccttc gggaacgtgg ttgacctaca caggtgccat caaattggat 1020
gacaaagatc caaatttcaa agatcaagtc attttgctga ataagcatat tgacgcatac 1080
aaa 1083
<210> 4
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15

Claims (9)

1. A fusion protein aiming at a novel coronavirus variant strain Delta, which is formed by fusing an S protein, an M protein and an N protein;
the nucleotide sequence of the coding gene of the S protein is shown as SEQ ID NO: 1 is shown in the specification;
the nucleic acid sequence of the coding gene of the M protein is shown as SEQ ID NO: 2 is shown in the specification;
the nucleotide sequence of the coding gene of the N protein is shown as SEQ ID NO: 3, respectively.
2. The fusion protein of claim 1, wherein the fusion protein is an S-M-N fusion protein.
3. The fusion protein of claim 2, further comprising a flexible linker between each two proteins;
the amino acid sequence of the flexible linker is (GGGGS) n, wherein n is an integer and is more than or equal to 1 and less than or equal to 4.
4. An intranasal vaccine against Delta variant strain of neocoronavirus, which comprises the fusion protein of claim 1 and an adjuvant.
5. The nasal spray vaccine according to claim 4, wherein the concentration of the fusion protein is 0.1-0.2 g/L;
6. the nasal spray vaccine of claim 4 or 5, wherein the adjuvant is PLGA.
7. The nasal spray vaccine of claim 6, wherein the final concentration of PLGA in the vaccine is 1-2 mg/mL.
8. Use of the fusion protein of any one of claims 1 to 3 for preparing a vaccine against Delta of a variant strain of a new coronavirus.
9. The use according to claim 8, wherein the vaccine is a nasal spray vaccine.
CN202111011970.8A 2021-08-31 2021-08-31 Fusion protein for Delta of new coronavirus variant strain, nasal spray type vaccine, and preparation method and application thereof Pending CN113621076A (en)

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