CN113151331A - SARS-ConV-2 virus S protein extramembranous BD end domain high targeting recombinant protein and subunit vaccine thereof - Google Patents
SARS-ConV-2 virus S protein extramembranous BD end domain high targeting recombinant protein and subunit vaccine thereof Download PDFInfo
- Publication number
- CN113151331A CN113151331A CN202110411059.XA CN202110411059A CN113151331A CN 113151331 A CN113151331 A CN 113151331A CN 202110411059 A CN202110411059 A CN 202110411059A CN 113151331 A CN113151331 A CN 113151331A
- Authority
- CN
- China
- Prior art keywords
- protein
- seq
- virus
- sars
- recombinant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a high targeting recombinant protein of BD end domain outside a SARS-ConV-2 virus S protein membrane and subunit vaccine thereof, wherein the nucleotide sequence of the encoded recombinant protein is shown as SEQ ID NO: 5 or as shown in SEQ ID NO: 6 is shown in the specification; the novel coronavirus subunit vaccine comprises the recombinant protein and a pharmaceutically acceptable adjuvant. The invention screens the surface antigenic determinant of the S protein extramembranous domain, finds the Sc and Se peptide segment gene sequences which can stimulate cells to generate strong immune reaction, carries out codon optimization, clones the gene sequences to a plasmid vector containing a diphtheria toxin T structure, secretes, expresses and purifies the gene sequences to obtain the purified recombinant SARS-ConV-2 virus S protein extramembranous BD end structural domain high-targeting recombinant protein, and the experimental verification shows that the recombinant protein can induce specific cell reaction, is safe and efficient, and has good development and application prospects.
Description
Technical Field
The invention belongs to the field of biotechnology, and relates to a BD-terminal domain high-targeting recombinant protein outside a SARS-ConV-2 virus S protein membrane and a subunit vaccine thereof.
Background
SARA-Cov-2 belongs to the genus of β -coronavirus in the family of coronaviridae, and belongs to the same genus as Severe Acute Respiratory Syndrome (SARS) coronavirus (SARS-CoV) and Middle East Respiratory Syndrome (MERS) coronavirus (MERS-CoV). SARA-Cov-2 is a positive-strand single-stranded RNA virus, the genome is 29.9kd, and for RNA viruses, the genome is large and encodes 9860 amino acids in total. The genome comprises a5 ' cap structure and a 3 ' Ploy a tail, the replicase genes encoding 16 non-structural proteins are close to the 5 ' end, accounting for about 2/3 of the whole genome, and the remaining 1/3 genome encodes 4 structural proteins. The known genomes of various coronaviruses were subjected to sequence alignment, and found to have homology of 54% at the whole genome level, and homology of encoding genes for structural proteins and non-structural proteins of 58% and 43%, respectively, indicating that the structural proteins are more diverse. The S protein of coronavirus is a type I virus fusion protein on the virosomal shell, and plays an important role in virus infection by recognizing a host cell surface receptor and mediating the fusion of the virus and a cell membrane. The S protein contains two subunits of S1 and S2, and researches show that SARS-Cov-2 has higher homology with the S protein S1 subunit on the surface of SARS-Cov virus in 2003, which is mainly reflected in that a receptor Binding Domain (BD) of SARS-Cov-2 has almost the same three-dimensional space structure as SARS-Cov, which indicates that the receptors bound by the two viruses are the same, namely bronchial epithelial cells and lung epithelial cells are infected through ACE2 receptors. Based on the key role of the S protein in SARS-Cov-2 virus infection, it can be the target of therapeutic drug design.
Although the mRNA vaccine is developed quickly, the complicated virus culture or recombinant protein expression process in vitro is avoided, only the synthesis of a specific mRNA sequence is needed, the mRNA is embedded by using liposome and then injected into human muscle tissue, and the mRNA enters into tissue cells to express pathogenic protein to trigger the immune emergency response of human body, but no vaccine can pass through clinical experiments due to various problems of the mRNA vaccine. For example: some people are sensitive to exogenous RNA and degrade without expression; some people are not sensitive and can express for a long time, so that antigen tolerance is caused, and antibodies cannot be generated; also, there are strong toxic and side effects due to intracellular expression of pathogenic antigens through interaction with intracellular proteins, etc. In clinical studies, these severe side effects include not only those at the injection site, but also systemic side effects. The research suggests that the mRNA vaccine is continuously expressed in vivo to generate antigen protein, possibly breaking the immune balance of the organism per se and triggering immune tolerance. In addition to potential side effects, some clinical studies have shown that mRNA vaccines are much less effective in humans than in animals, indicating that there is a need to further study the differences in response to mRNA vaccines from animal to human, to the extent that which immune pathways are most effective in humans? Can the efficacy be improved by altering the immunostimulatory expression of the vaccine? These are problems to be solved, and mRNA vaccines have a long way to go.
In contrast to mRNA vaccines, recombinant protein subunit vaccines also have certain advantages in some respects. Firstly, the required equipment is simple, the large-scale production is easy, the steps of inactivation or attenuation of strains and the like are avoided, and the safety is high; secondly, the recombinant protein vaccine belongs to active immunity, has long-acting prevention effect and is a perfect supplement to a therapeutic mRNA vaccine; in addition, the recombinant protein vaccine does not have the problem of continuous expression in vivo like mRNA vaccine, and cannot cause immune tolerance; moreover, compared with mRNA vaccine, the recombinant protein vaccine has longer shelf life and is more beneficial to strategic storage; finally, recombinant protein subunit vaccines have only a few major surface proteins, avoiding the production of many unrelated antigen-induced antibodies, thereby reducing the side effects of the vaccine and associated diseases caused by the vaccine.
With the development of biotechnology, recombinant protein vaccines have also entered the precise era. The most important essence of recombinant protein vaccines is that the protein to be recombined can accurately and efficiently elicit an immune emergency response against exogenous bacteria or viruses, and therefore, the selection of antigens for use as vaccines is of great importance. It is more desirable that the recombinant protein vaccine only induce in vivo the production of antibodies against surface antigens of pathogenic bacteria or viruses, so that the immune system can move around less frequently and efficiently and accurately eliminate the pathogenic bacteria or viruses. Therefore we need to select the envelope or capsid protein of the outermost layer of pathogenic bacteria or virions as the immunogen. The proteins are often membrane proteins, so that the membrane inner parts of the proteins need to be removed, even in order to generate the targeted antibodies more accurately and efficiently, the antigenic determinants of the membrane outer parts of the proteins are directly extracted to be used as immunogens, the antigenic determinants in the protein molecular structures are also knocked out, and only the antigenic determinants on the surfaces of the protein molecules are reserved.
The multi-target recombinant protein vaccine adopts fusion expression of a plurality of special antigenic determinant sequences, and is not complete pathogen protein, so that rapid toxic and side effects cannot be generated, and the multi-target recombinant protein vaccine has high safety. In addition, the antigenic determinant of the multi-target recombinant protein vaccine should be selected as much as possible to select Th target sequences, so that the killer T cells of the human body can be effectively activated, and pathogens can be further killed. Moreover, the multi-target recombinant protein vaccine is a model vaccine, the structural model of the vaccine is stable and consistent, and the vaccine consists of an immune strong activation region and an antigenic determinant region, so that the vaccine can be free from clinic. Finally, as a model vaccine, the multi-target recombinant protein vaccine is easy to be produced in a standardized way, and a standard factory building for small-scale production is established.
The production technology of the recombinant protein vaccine is mature, and a plurality of remarkable achievements are achieved in the fields of medicine and veterinary medicine, for example, the human papilloma virus recombinant protein vaccine, the anti-tumor recombinant protein vaccine, the taenia solium recombinant vaccine and the like are on the market. With the development and the intensive research of our project, the research of the 2019-nCoV virus and SARS virus double-effect multi-target recombinant protein vaccine is determined to be successful.
Therefore, there is a need to develop a new effective vaccine against a novel coronavirus.
Disclosure of Invention
In order to solve the technical problem, the invention provides a BD end domain high-targeting recombinant protein outside a SARS-ConV-2 virus S protein membrane and subunit vaccine, wherein the recombinant protein can induce specific cell reaction.
In the first aspect of the invention, a BD end domain high targeting recombinant protein outside a SARS-ConV-2 virus S protein membrane is provided, and a nucleotide sequence for coding the recombinant protein is shown as SEQ ID NO: 5 or as shown in SEQ ID NO: and 6.
In a second aspect of the invention, there is provided a nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO: 5 or SEQ ID NO: and 6.
In the third aspect of the invention, a recombinant expression vector is provided, the recombinant expression vector can express the SARS-ConV-2 virus S protein extramembranous BD end domain high-targeting recombinant protein, and the recombinant expression vector contains the nucleic acid molecule.
Further, the recombinant expression vector includes one of an expression plasmid vector, a phage vector, and a viral vector.
Further, the nucleotide sequence of the expression region of the expression plasmid vector is shown as SEQ ID NO: 5 or as shown in SEQ ID NO: and 6.
Further, the expression plasmid vectors are pET28a-DTT-2Sc and pET28a-DTT-3 Se.
In a fourth aspect of the invention, there is provided an engineered bacterium or an engineered cell comprising any one of the recombinant expression vectors.
In the fifth aspect of the invention, a method for preparing a BD end domain high targeting recombinant protein outside the S protein membrane of SARS-ConV-2 virus is provided, which comprises the following steps:
obtaining antigenic determinants Sc and Se of the BD end structural domain outside the SARS-ConV-2 virus S protein membrane, wherein the nucleotide sequences of the Sc are respectively shown as SEQ ID NO: 1, and the nucleotide sequence of Se is shown as SEQ ID NO: 2 is shown in the specification;
constructing and obtaining the Sc and Se expression plasmid, wherein the nucleotide sequence of the Sc expression plasmid expression region is shown as SEQ ID NO: 5, the nucleotide sequence of the expression region of the Se expression plasmid is shown as SEQ ID NO: 6 is shown in the specification;
and respectively carrying out secretory expression and purification on the Sc and Se expression plasmids to obtain the purified recombinant SARS-ConV-2 virus S protein extramembranous BD end domain high-targeting recombinant protein.
In a sixth aspect of the invention, a novel coronavirus subunit vaccine is provided, which comprises the recombinant protein and a pharmaceutically acceptable adjuvant.
Further, the adjuvant comprises at least one of aluminium hydroxide, lecithin, Freund's adjuvant, MPL TM, IL-12, aluminium hydroxide combined CpG ODN composite adjuvant, ISA51VG, ISA720VG, MF59, QS21 and AS03 adjuvants.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
the invention provides a nano antibody for resisting SARS-COV-2 virus S protein RBD structural domain;
the invention provides a SARS-ConV-2 virus S protein extramembranous BD end domain high targeting recombinant protein, a recombinant expression vector, an engineering bacterium or an engineering cell and a subunit vaccine, and the method of the invention utilizes the homology of SARS-Cov-2 and SARS-Cov virus S protein to screen the surface antigenic determinant of the extramembranous domain of the S protein, and finds a sequence which can stimulate cells to generate strong immune response. Specifically, Se and Sc peptide segment gene sequences are cloned to a plasmid vector containing a diphtheria toxin T structure and subjected to codon optimization, then secretion expression and purification are carried out, and the purified recombinant SARS-ConV-2 virus S protein membrane outer N-end structural domain high-targeting recombinant protein is obtained.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1: a three-dimensional model of SARA-Cov-2 virus S protein; a is the amino acid comparison of S proteins of SARA-Cov-2 virus and SARS-Cov virus; b is a three-dimensional structure model of the spinous process formed by 3S proteins of SARA-Cov-2 virus; c is a three-dimensional structure model of SARA-Cov-2 virus S protein;
FIG. 2: potential antigenic determinants (arranged from high to low according to a score) of the BD end Domain outside the S protein membrane of the SARA-Cov-2 virus on Domain I/II;
FIG. 3: a design schematic diagram of recombinant protein plasmids of pET28a-DTT-Sc and pET28 a-DTT-Se;
FIG. 4: the purification of DTT-2Sc and DTT-3Se proteins is analyzed by 15 percent SDS-PAGE;
FIG. 5: antiserum reaction; NC1 incubation with PBS only; NC2 incubation with microspheres only; 2Sc, 3 Se: incubation with antiserum cleaned with DTT-beads only; DTT-2Sc, DDT-3 Se: incubate with unclean antiserum only;
FIG. 6: detecting cell reaction triggered by DTT-2Sc and DDT-3Se vaccines; stimulating spleen cells by using DTT-2Sc and DDT-3Se 7 days after the mice are immunized for the third time, and detecting cell proliferation by using a CCK8 kit;
FIG. 7: detecting cell reaction triggered by DTT-2Sc and DDT-3Se vaccines; 7 days after the third immunization of the mice, spleen cells are stimulated by DTT-2Sc and DDT-3Se, effector cells with a certain proportion are added for co-culture, and cell proliferation is detected by an LDH cytotoxicity detection kit.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
In order to solve the technical problems, the embodiment of the invention provides the following general ideas:
according to a typical embodiment of the present invention, a SARS-ConV-2 virus S protein extracellular BD-terminal domain high targeting recombinant protein is provided, wherein a nucleotide sequence encoding the recombinant protein is shown in SEQ ID NO: 5 or as shown in SEQ ID NO: and 6.
The method analyzes single S protein by constructing a three-dimensional structure model of SARS-ConV-2 virus S protein, and determines that the extramembranous domain is a target site for research and development of antibodies and vaccines. And (2) analyzing the antigenic determinants of the BD end domain outside the SARS-ConV-2 virus S protein film to obtain the antigenic determinants Sc and Se of the BD end domain outside the SARS-ConV-2 virus S protein film, wherein the amino acid sequences of the Sc are respectively shown as SEQ ID NO: 3, and the amino acid sequence of Se is shown as SEQ ID NO: 4 is shown in the specification;
in order to further improve the immune stress capability of ADn, a diphtheria toxin T structural Domain (DTT) is coupled to the N terminal of ADn, the structural domain is already used for producing vaccines and has strong capability of activating human immune stress response, and the surface of the structural domain has a plurality of antigenic determinants which can activate human CD4 and CD 8T cells and can effectively activate the virus elimination effect of human killer T cells. Specifically, peptide gene sequences of Sc and Se are synthesized, cloned to a pET-28a plasmid vector containing a diphtheria toxin T structure, pET28a-DTT-Sc and pET28a-DTT-Se plasmids are constructed, DTT is a diphtheria toxin T structure domain, and non-immunogenic GS Linker is added between DTT and Sc and Se: the nucleotide sequence is GGTGGTGGTGGTAGTGGTGGTGGTGGTAGT-3'.
In order to facilitate the soluble expression of the tandem polypeptide, the nucleic acid sequence encoding the antigen peptide fragment needs to be codon-optimized for expression in Escherichia coli. The optimized plasmids are named as pET28a-DTT-2Sc and pET28a-DTT-3Se (figure 3); the nucleotide sequence of the expression region of the Sc expression plasmid is shown as SEQ ID NO: 5, the nucleotide sequence of the expression region of the Se expression plasmid is shown as SEQ ID NO: 6 is shown in the specification;
and carrying out secretory expression and purification on the plasmid to obtain the purified recombinant SARS-ConV-2 virus S protein extramembranous BD end domain high-targeting recombinant protein.
In conclusion, the invention establishes a preparation method of the novel SARS-Cov-2 virus S protein extramembranous BD end domain high-targeting recombinant vaccine by developing protein expression and inducing animal immunity on the basis of fully analyzing the coronavirus S protein extramembranous domain and the latest research result of the recombinant protein vaccine, and the method is suitable for preparing the novel genetic engineering recombinant protein vaccine, is safe and efficient, and has good development and application prospects.
According to another exemplary embodiment of the present embodiments, there is provided a nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO: 5 or SEQ ID NO: and 6.
Biological materials containing the nucleic acid molecule are also within the scope of the present invention, including one of recombinant DNA, plasmid vectors, phage vectors, and viral vectors.
According to another exemplary embodiment of the present invention, a recombinant expression vector is provided, wherein the recombinant expression vector is capable of expressing the SARS-ConV-2 virus S protein extramembranous BD end domain high-targeting recombinant protein, and the recombinant expression vector contains the nucleic acid molecule. The recombinant expression vector comprises one of an expression plasmid vector, a phage vector and a viral vector. In the embodiment of the invention, the nucleotide sequence of the expression region of the expression plasmid vector is shown as SEQ ID NO: 5 or as shown in SEQ ID NO: and 6. The expression plasmid vector is pET28a-DTT-2Sc and pET28a-DTT-3 Se.
According to another exemplary embodiment of the embodiments of the present invention, an engineered bacterium or an engineered cell comprising the recombinant expression vector is provided. The engineering bacteria or the engineering cells can be suspension cells, and mammalian cell strains of human vaccines such as CHO series and 293, 293FT and the like are all within the protection scope of the invention.
According to another exemplary embodiment of the present invention, there is provided a method for preparing a BD-terminal domain highly targeted recombinant protein outside the S protein membrane of SARS-ConV-2 virus, the method comprising:
obtaining antigenic determinants Sc and Se of the BD end structural domain outside the SARS-ConV-2 virus S protein membrane, wherein the nucleotide sequence of the Sc is shown as SEQ ID NO: 1, and the nucleotide sequence of Se is shown as SEQ ID NO: 2 is shown in the specification;
constructing and obtaining the Sc and Se expression plasmid, wherein the nucleotide sequence of the Sc expression plasmid expression region is shown as SEQ ID NO: 5, the nucleotide sequence of the expression region of the Se expression plasmid is shown as SEQ ID NO: 6 is shown in the specification;
and respectively carrying out secretory expression and purification on the Sc and Se expression plasmids to obtain the purified recombinant SARS-ConV-2 virus S protein extramembranous BD end domain high-targeting recombinant protein.
According to another exemplary embodiment of the present invention, there is provided a novel coronavirus subunit vaccine comprising said recombinant protein and a pharmaceutically acceptable adjuvant.
The adjuvant comprises at least one of aluminium hydroxide, lecithin, Freund's adjuvant, MPL TM, IL-12, aluminium hydroxide combined CpG ODN composite adjuvant, ISA51VG, ISA720VG, MF59, QS21 and AS03 adjuvants. In other embodiments, the adjuvant may be in other forms.
The novel coronavirus subunit vaccine can be prepared into nasal drops, sprays and intramuscular injections.
The subunit vaccine is applied, and the result of the immunized mouse shows that the DTT-2Sc and DTT-3Se vaccines can induce specific cell reaction.
The effects of the present application will be described in detail below with reference to examples and experimental data. The specific experimental conditions and methods not indicated in the following examples are generally in accordance with conventional conditions such as: J. SummBruk et al, science publishers, 1992, molecular cloning, A laboratory Manual (third edition); l. speekt et al, scientific press, 2001, cell protocols, etc., or as recommended by the manufacturer.
Example 1 analysis and design of antigenic determinants of BD-terminal extramembranous domain of S protein of SARS-Cov-2 Virus
1. Construction of three-dimensional model of SARA-Cov-2 virus S protein
The amino acid sequences of the S proteins of two coronaviruses, SARA-Cov-2 and SARS-Cov, were retrieved from the national center for bioinformatics technology database (https:// www.ncbi.nlm.nih.gov), with the accession numbers QHD43416 and P59594 in GenBank, respectively. Comparing the amino acid sequences of SARS-Cov-2 and SARS-Cov S protein by Clustal software, the amino acid uniformity is 76%, and the homology is higher. The highly homologous sequences of the S proteins of both viruses are mainly centered in the middle to the C-terminus. Further constructing a three-dimensional structure model of the 2019-nCoV virus S protein. The 2019-nCoV virus forms a spike structure on the surface of a virus particle envelope through polymerization of 3S proteins. Further analysis of the structure of the single S protein revealed that Domain I and Domain II of the S protein are the membrane-external portions of the spike structure, while Domain III is the membrane-internal portion, and thus are the target sites for antibody and vaccine development. (FIG. 1A, B)
The antigenic determinants of Domain I and Domain II of the 2019-nCoV virus S proteins were further resolved. We predicted 24 potential antigenic determinants, and among them, peptides No. 2, 3, 5, 6, 9, 11, 14, 15, 18, 20, 21, 23 and 24, and 13 in total, all located on the surface of Domain I or II molecules, are potential antibody recognition sites. Comparing these antigen recognition sites with the sequence of SARS S protein, it is found that peptide segments No. 3, 11, 14, 21 and 23, and 5 peptide segments in total have high homology with SARS S protein, therefore, these 5 peptide segments are the key antigenic determinant sites for the development of 2019-nCoV virus and SARS virus immunological type detection reagent and vaccine. (FIG. 2)
2. Designing SARS-Cov-2S protein BD end structural domain antigen determinant
The antigenic determinants of the BD-terminal domain of the S protein were further resolved. We predict 10 potential antigenic determinants, and the peptides No. 1, 4, 6 and 9 are all located in the protein structure and cannot form powerful binding sites, so that the potential antigenic determinants are not considered. The No. 2 peptide fragment is positioned outside a BD terminal protein molecule and at the bottom of a binding site of a receptor and a ligand, and can be used as a candidate antigen target, but further verification is needed, and the No. 8 peptide fragment is positioned at the middle part and the inner part of a BD structure domain, so that the potential of serving as the antigen target is weak. The No. 3 peptide segment is positioned at the positive position of a receptor-ligand binding site on the surface of BD terminal molecules and is named as Sc, and the No. 5 peptide segment contains 6 amino acids, is positioned at the bottom side of the receptor-ligand binding site and is positioned at the outer side when the receptor binding domain is in a closed or open state and is named as Se. The Sc peptide and the Se peptide are respectively used as molecular targets of BD terminal antigenic determinants outside the S protein membrane. Meanwhile, in order to further improve the immune stress capability of the target spot, the N end of the antigen peptide segment target spot is connected with a diphtheria toxin T structure (DTT), the diphtheria toxin T structure is applied to a vaccine rope factory and has strong capability of activating human immune stress response, and the surface of the diphtheria toxin structure has a plurality of antigenic determinants for activating human CD +4 and CD +8T cells, so that the killing T cells of the human body can be effectively activated to remove viruses. (FIG. 2)
3. Construction of gene expression plasmid pET-28a-DTT containing SARS-Cov-2S protein BD end antigen peptide segment
Synthesizing the Sc and Se peptide segment gene sequences analyzed in the step 2, and cloning the Sc and Se peptide segment gene sequences to a pET-28a plasmid vector containing a diphtheria toxin T structure, wherein the gene sequences are as follows:
(1) sc, nucleotide sequence shown as SEQ ID NO: 1 is shown in the specification; the amino acid sequence is shown as SEQ ID NO: 3 is shown in the specification;
(2) se, nucleotide sequence shown as SEQ ID NO: 2 is shown in the specification; the amino acid sequence is shown as SEQ ID NO: 4 is shown in the specification;
4. pET28a-DTT-Sc and pET28a-DTT-Se plasmids are constructed, DTT is a T structural domain of diphtheria toxin, and non-immunogenic GS Linker is added between DTT, Sc and Se. In order to facilitate the soluble expression of the tandem polypeptide, the nucleic acid sequence encoding the antigen peptide fragment needs to be codon-optimized for expression in Escherichia coli.
(1) The specific steps for constructing the pET28a-DTT-2Sc plasmid are as follows:
the pET28a vector is cut by restriction enzymes Nde I and BamH I (Thermo Fisher), the DTT gene is cut by Nde I and BamH I (Thermo Fisher), then the cut vector and DTT gene are respectively recovered by agarose gel electrophoresis, and then linked by T4 ligase (New England Biolabs), and then transformed into competent DH5, and the correct clone is selected and identified, thus obtaining pET28a-DTT vector;
the pET28a-DTT vector is cut by restriction endonucleases BamH I and HindIII (thermo Fisher), Sc gene (containing GS Linker) is cut by BamH I and HindIII (thermo Fisher), then the cut vector and Sc gene are respectively recovered by agarose gel electrophoresis, and are linked by T4 ligase (New England Biolabs), and then are transformed into competence DH5, and the correct clone is selected and identified, thus obtaining pET28a-DTT-2Sc expression plasmid. The optimized plasmid was named pET28a-DTT-2Sc (FIG. 3); the nucleotide sequence of pET28a-DTT-2Sc is shown in SEQ ID NO: 5, respectively.
(2) The specific steps of the pET28a-DTT-3Se plasmid construction are as follows:
the pET28a vector is cut by restriction enzymes Nde I and BamH I (Thermo Fisher), the DTT gene is cut by Nde I and BamH I (Thermo Fisher), then the cut vector and DTT gene are respectively recovered by agarose gel electrophoresis, and then linked by T4 ligase (New England Biolabs), and then transformed into competent DH5, and the correct clone is selected and identified, thus obtaining pET28a-DTT vector;
the pET28a-DTT vector is cut by restriction endonucleases BamH I and HindIII (thermo Fisher), the Se gene (containing GS Linker) is cut by BamH I and HindIII (thermo Fisher), then the cut vector and the Se gene are respectively recovered by agarose gel electrophoresis, then T4 ligase (New England Biolabs) is used for linking, and then the vector and the Se gene are transformed into competence DH5, and the correct clone is selected and identified, thus obtaining the pET28a-DTT-3Se expression plasmid. The optimized constructed plasmid was named pET28a-DTT-3Se (FIG. 3); the nucleotide sequence of pET28a-DTT-3Se is shown as SEQ ID NO: and 6.
Example 2 expression and purification of related antigenic determinant fragments at the BD end of the S protein
1. Expression of 2Sc and 3Se
The constructed plasmids were transformed into BL21(DE3) expression competent cells, respectively, plated on a Carna-resistant LB plate, and cultured overnight in a 37 ℃ incubator to grow a single clone. Each of 4 monoclonals was selected and cultured in 5ml of kanamycin-resistant LB medium at 200rpm, 37 ℃ for 5 hours. When the OD600 reached 0.6-0.8, 500. mu.L of the sample was taken out and placed in a new 1.5mL EP tube and labeled before induction. The remaining bacterial solution was induced by adding IPTG (final concentration of 1mM), and the samples marked before and after induction were incubated at 37 ℃ for 4 hours. The bacterial suspension before and after induction was aspirated out of 200. mu.L each at 12000rpm, and centrifuged for 5 min. The supernatant was removed, 50. mu.L of PBS was added for resuspension, 2-loading buffer was added and mixed well, and the mixture was boiled in boiling water for 10 min. Electrophoretic analysis was performed by 15% SDS-PAGE.
Two high-expression positive clones were selected and inoculated in 100mL of LB medium containing kanamycin antibiotic and cultured at 180rpm at 37 ℃ overnight. The next day, 100mL of the bacterial suspension was inoculated into 2L of LB medium, the temperature was adjusted to 30 ℃ and the culture was expanded. When the culture is carried out until the OD600 is 0.6-0.8, 0.4mL of 500mmol/L IPTG is added, the temperature is adjusted to 16 ℃, and the culture is carried out overnight at 160 rpm.
Pouring the bacterial liquid into a cleaned 1mL centrifuge tube, balancing, centrifuging at 5000rpm for 20min, discarding the supernatant, and collecting the thallus to a 50mL centrifuge tube. 30mL of Buffer A (25mM Tris,500mM NaCl, 10% (v/v) Glycerol, pH7.5) was added to resuspend the cells, the cells were centrifuged at 5000rpm for 15min, the supernatant was discarded, 50mL of Buffer A was added to resuspend the cells, and the resuspended cells were placed at-80 ℃ overnight.
2. Purification of 2Sc, 3Se protein
1) Respectively taking out the collected bacterial liquid from a refrigerator at minus 80 ℃, and placing the bacterial liquid in a water bath for dissolving for 30min until the bacterial liquid is dissolved safely. Pouring the dissolved bacterial liquid into a beaker, placing the beaker in ice water for mixing, and carrying out ultrasonic crushing under the ultrasonic conditions: the ultrasonic treatment is carried out for 5s, the rest time is 10s, the power is 40 percent, the volume of each ultrasonic treatment is not more than 50mL, and the total ultrasonic treatment time is 60 min.
2) And (3) putting the lysate after ultrasonic treatment into a precooled centrifuge, centrifuging at 6000rpm for 20min at 4 ℃, performing suction filtration on the supernatant by using a 0.45-micron filter membrane, pouring the filtrate into a beaker, and placing the beaker on ice. The precipitate and the supernatant were separately taken and placed in a 1.5mL EP tube.
3) The supernatant was affinity purified using a Ni prepacked column, first 10 column volumes were equilibrated with Buffer A (25mM Tris,500mM NaCl, 10% (v/v) Glycerol, pH7.5) at a flow rate of 3mL/min using a 5mL Ni column, then the protein after suction filtration was loaded at a flow rate of 2mL/min, and after loading was complete, the column volumes were at least 10. Then, different imidazole concentrations were set with Buffer B (50mM Tris-HCl, 500mM imidazole, pH7.0), and elution was performed at 25mM, 125mM, 250mM, and 500mM, respectively, at a flow rate of 2mL/min, and collection was performed until absorbance did not change any more at the time when absorbance started to increase at each gradient. When the absorbance was constant at the elution of 500mM imidazole, the elution was terminated.
4) The protein samples eluted at different imidazole concentrations were separately loaded into 1.5mL EP tubes for 15% SDS-PAGE analysis. Protein samples of the expected size and low impurity content were subjected to Q pre-column purification by first equilibrating a 5mL Q column with Buffer C (25mM Tris, pH7.5) at a flow rate of 3mL/min for 10 column volumes. Diluting the pre-purified protein sample by 4 times by using Buffer C, then loading the diluted protein with the flow rate controlled to be 2mL/min, and washing impurities by using the Buffer C after loading is finished, wherein at least 10 column volumes are used. Then, Buffer D (25mM Tris, 1M NaCl, pH7.5) was used to set different NaCl concentrations, 25mM, 75mM, 125mM, 250mM, 500mM were used for elution, respectively, at a flow rate of 2mL/min, and collection was performed until absorbance did not change any more at the time when absorbance started to rise at each gradient. When the absorbance was constant at the elution with 500mM NaCl, the elution was terminated.
5) The protein samples eluted with different NaCl concentrations were respectively put into 1.5mL EP tubes for 15% SDS-PAGE electrophoretic analysis. Proteins consistent with the expected size and free of carryover were concentrated by ultrafiltration using a 3kD concentrator tube. Subpackaging the obtained protein 1 mL/tube, quick freezing with liquid nitrogen, and storing at-80 deg.C (FIG. 4)
Example 3 functional assay of the BD-terminal Domain highly Targeted recombinant protein vaccine outside the SARS-Cov-2S protein Membrane
In order to evaluate the immune efficacy of the BD end domain high targeting recombinant protein vaccine outside the SARS-Cov-2S protein membrane, the invention adopts a BALB/c mouse as an animal model.
1. SARS-Cov-2S protein membrane outer BD end domain high target recombinant protein vaccine immunity
1) SPF grade 5-6 week old healthy female BALB/c mice were purchased at 8 and randomly divided into 2 groups of 4 mice each, 2Sc, 3Se as experimental groups.
2) Preparing a vaccine: each mouse received 50. mu.g of antigen protein and 300. mu.g of Alum, and 30. mu.g of CpG, in a total volume of 200. mu.L each, the volume of antigen protein, Alum, and CpG was first calculated based on protein concentration, and then Al (OH)3The adjuvant, the antigen and the CpG are mixed evenly and placed on ice for 15min, and the vaccine injection is completed within 2 hours as far as possible.
3) Vaccine immunization injection: injecting four needles under the skin at abundant positions of axillary lymph nodes on days 1, 12 and 21 for immunization, wherein the dosage of each needle is 50 mu L, and the total dosage is 200 mu L;
4) collecting serum: blood is taken from an orbit, an eyeball is taken by using a sterile elbow forceps holder, a thumb and a forefinger are twisted, the blood vertically flows into a 1.5mL sterile centrifuge tube from the orbit at different speeds, meanwhile, the heart of the mouse is lightly pressed by the middle finger of the left hand to accelerate the blood pumping speed of the heart, and when the blood runs out, the mouse is killed by a dislocation method. Each mouse collected 100. mu.L of blood, and cross contamination was avoided. And (3) placing the collected serum at room temperature for 2h, placing the serum in a refrigerator at 4 ℃ for 3-4 h, and then centrifuging at 4000rpm for 10min, thus finishing centrifugation. The agglutinated clot was seen at the bottom and clear serum was above, the supernatant was quickly aspirated and aliquoted (10 μ L per tube) and frozen at-20 ℃ for use.
2. Efficacy evaluation of BD end domain high targeting recombinant protein vaccine outside SARS-Cov-2S protein membrane
1) Antiserum detection
Antibody titers in mouse sera were determined by ELISA. The method comprises the following specific steps:
microsphere neutralizing antibody: in order to detect specific antibodies against the BD-terminal extramembranous domain of the S protein, the BD-terminal domain 2Sc and 3Se of the S protein without carrier protein should be used as detection protein, so in order to exclude antibodies against DTT in serum of DTT-2Sc and DTT-3Se proteins of experimental groups, antiserum of DTT-2Sc and DTT-3Se immunized mice is firstly combined with DTT-Sn1 antigen (nucleotide sequence encoding DTT-Sn1 antigen is shown as SEQ ID NO: 7), the antibodies combined with DTT in serum are absorbed, then the serum is sucked out and transferred to the holes of DTT-2Sc and DTT-3Se protein planking, and the antibodies against 2Sc and 3Se in serum are detected.
Coating: diluting 2Sc and 3Se proteins to 1 μ g/ml with a buffer solution (0.05M carbonate buffer solution of pH 9.6), coating a 96-well ELISA plate (product of NEST Co., Ltd., the same below) with 100 μ l per well, and coating overnight at 4 ℃;
and (3) sealing: washing ELISA plate coated with DTT-2Sc and DTT-3Se antigens with PBS-T (PBS containing 0.1% Tween-20, pH7.5, the same below) for 6 times, each for 5 min; adding 100 μ l blocking solution (PBST containing 3% BSA), blocking at 37 deg.C for 60 min; PBST was washed 5 times.
Adding serum to be detected: diluting mouse serum neutralized by microspheres with PBST containing 1% BSA at a 1:100 gradient, adding the diluted serum into each well (100. mu.l/well) of an ELISA plate, and incubating at 37 ℃ for 60 min; PBST was washed 5 times.
Adding an enzyme-labeled secondary antibody: the secondary antibody is horse radish peroxidase labeled goat anti-mouse IgG antibody (product of ABClonal company), diluted 1:5000 with 1% BSA-containing PBST, added into each well (100. mu.l/well) of ELISA plate, and acted at 37 ℃ for 60 min; PBST was washed 5 times.
Addition of revealing substrate solution: a chromogenic background solution (containing 0.075% H) was added to each well of the microplate2O21mg/ml of tetramethylbiphenyl, TMB), 100 mul per hole, developing for 15min at normal temperature; 2M H per well2SO4The reaction was stopped with 50. mu.l.
OD450Value determination: the optical density absorption (OD) in each well was measured at a wavelength of 450nm on a microplate reader450)
The result shows that the high targeting recombinant vaccine immunization of the BD end domain outside the S protein membrane prepared by the invention can induce the organism to generate high-level specific antibody (figure 5).
2) Cell proliferation assay
In vitro stimulation of lymphocytes to see if they can proliferate specifically for the 2Sc and 3Se antigens, each group of spleen cells obtained should be stimulated with DTT that does not contain the carrier protein, so we can detect the proliferation of cells specific for 2Sc and 3Se by using 2Sc and 3Se proteins after microsphere neutralization.
Splenocytes were isolated from the spleen of immunized mice and treated with ACK lysis buffer. The cells were cultured at 1X 105Cell/well concentration was plated in 96-well plates, 100. mu.L RPMI-1640 medium containing 10% FBS was added, stimulated with 30. mu.g/mL of the corresponding stimulating protein, and placed at 37 ℃ in 5% CO2The culture is carried out in a constant temperature and humidity incubator. After 72 hours, the cells were treated with CCK-8 solution according to the product instructions. The Optical Density (OD) at a wavelength of 450nm was measured, with the OD at 650 as a reference value. The Stimulation Index (SI) was calculated as the ratio of the Optical Density (OD) at 450nm of stimulated versus unstimulated cells. (FIG. 6)
The results show that significant cell proliferation was observed in spleen cells of mice treated with blocked DTT-2Sc and DTT-3Se, with stimulation indices slightly higher than those of PBS and microsphere groups and slightly lower than those of unblocked DTT-2Sc and DTT-3Se groups.
3) Cytotoxicity test
And further detecting the killing effect of the stimulated spleen cells on target cells as effector cells. Firstly, transfecting a BD domain plasmid pcDNA4-S (BD) of an S protein into an A549 cell, carrying out endogenous transient expression, fixing by using methanol after 24h of expression, and carrying out osmotic perforation on a cell membrane by using Triton X-100 to establish a target cell. And finally, adding spleen cells of mice immunized by the DTT-2Sc and the DTT-3Se according to a certain proportion with the target cells for co-culture.
In conclusion, the results show that the DTT-2Sc and DTT-3Se vaccines can induce specific cell responses.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Sequence listing
<110> Wuhan university
<120> SARS-ConV-2 virus S protein extramembranous BD end domain high targeting recombinant protein and subunit vaccine thereof
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 246
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ggttttaatt gttactttcc tttacaatca tatggtttcg gtggtggtgg tagtggtggt 60
ggtggtagtg gttttaattg ttactttcct ttacaatcat atggtttcgg tggtggtggt 120
agtggtggtg gtggtagtgg ttttaattgt tactttcctt tacaatcata tggtttcggt 180
ggtggtggta gtggtggtgg tggtagtggt tttaattgtt actttccttt acaatcatat 240
ggtttc 246
<210> 2
<211> 150
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tgcccttttg gtgaagtttt taacgccacc ggtggtggtg gtagtggtgg tggtggtagt 60
tgcccttttg gtgaagtttt taacgccacc ggtggtggtg gtagtggtgg tggtggtagt 120
tgcccttttg gtgaagtttt taacgccacc 150
<210> 3
<211> 82
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gly Gly Gly
1 5 10 15
Gly Ser Gly Gly Gly Gly Ser Gly Phe Asn Cys Tyr Phe Pro Leu Gln
20 25 30
Ser Tyr Gly Phe Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Phe
35 40 45
Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gly Gly Gly Gly Ser
50 55 60
Gly Gly Gly Gly Ser Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr
65 70 75 80
Gly Phe
<210> 4
<211> 50
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Gly Gly
20 25 30
Gly Gly Ser Gly Gly Gly Gly Ser Cys Pro Phe Gly Glu Val Phe Asn
35 40 45
Ala Thr
50
<210> 5
<211> 757
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
catatgataa atcttgattg ggatgtcata agggataaaa ctaagacaaa gatagagtct 60
ttgaaagagc atggccctat caaaaataaa atgagcgaaa gtcccaataa aacagtatct 120
gaggaaaaag ctaaacaata cctagaagaa tttcatcaaa cggcattaga gcatcctgaa 180
ttgtcagaac ttaaaaccgt tactgggacc aatcctgtat tcgctggggc taactatgcg 240
gcgtgggcag taaacgttgc gcaagttatc gatagcgaaa cagctgataa tttggaaaag 300
acaactgctg ctctttcgat acttcctggt atcggtagcg taatgggcat tgcagacggt 360
gccgttcacc acaatacaga agagatagtg gcacaatcaa tagctttatc gtctttaatg 420
gttgctcaag ctattccatt ggtaggagag ctagttgata ttggtttcgc tgcatataat 480
tttgtagaga gtattatcaa tttatttcaa gtagttcata attcgtataa tcgtcccgcg 540
ggtggtggtg gtagtggtgg tggtggatcc ggttttaatt gttactttcc tttacaatca 600
tatggtttcg gtggtggtgg tagtggtggt ggtggtagtg gttttaattg ttactttcct 660
ttacaatcat atggtttcgg tggtggtggt agtggtggtg gtggtagtgg ttttaattgt 720
tactttcctt tacaatcata tggtttctaa caagctt 757
<210> 6
<211> 730
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
catatgataa atcttgattg ggatgtcata agggataaaa ctaagacaaa gatagagtct 60
ttgaaagagc atggccctat caaaaataaa atgagcgaaa gtcccaataa aacagtatct 120
gaggaaaaag ctaaacaata cctagaagaa tttcatcaaa cggcattaga gcatcctgaa 180
ttgtcagaac ttaaaaccgt tactgggacc aatcctgtat tcgctggggc taactatgcg 240
gcgtgggcag taaacgttgc gcaagttatc gatagcgaaa cagctgataa tttggaaaag 300
acaactgctg ctctttcgat acttcctggt atcggtagcg taatgggcat tgcagacggt 360
gccgttcacc acaatacaga agagatagtg gcacaatcaa tagctttatc gtctttaatg 420
gttgctcaag ctattccatt ggtaggagag ctagttgata ttggtttcgc tgcatataat 480
tttgtagaga gtattatcaa tttatttcaa gtagttcata attcgtataa tcgtcccgcg 540
ggtggtggtg gtagtggtgg tggtggatcc tgcccttttg gtgaagtttt taacgccacc 600
ggtggtggtg gtagtggtgg tggtggtagt tgcccttttg gtgaagtttt taacgccacc 660
ggtggtggtg gtagtggtgg tggtggtagt tgcccttttg gtgaagtttt taacgccacc 720
taacaagctt 730
<210> 7
<211> 706
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
catatgataa atcttgattg ggatgtcata agggataaaa ctaagacaaa gatagagtct 60
ttgaaagagc atggccctat caaaaataaa atgagcgaaa gtcccaataa aacagtatct 120
gaggaaaaag ctaaacaata cctagaagaa tttcatcaaa cggcattaga gcatcctgaa 180
ttgtcagaac ttaaaaccgt tactgggacc aatcctgtat tcgctggggc taactatgcg 240
gcgtgggcag taaacgttgc gcaagttatc gatagcgaaa cagctgataa tttggaaaag 300
acaactgctg ctctttcgat acttcctggt atcggtagcg taatgggcat tgcagacggt 360
gccgttcacc acaatacaga agagatagtg gcacaatcaa tagctttatc gtctttaatg 420
gttgctcaag ctattccatt ggtaggagag ctagttgata ttggtttcgc tgcatataat 480
tttgtagaga gtattatcaa tttatttcaa gtagttcata attcgtataa tcgtcccgcg 540
ggtggtggtg gtagtggtgg tggtggatcc tttcttgttt tattgccact agtctctagt 600
cagtgtgtta atcttacagg tggtggtggt agtggtggtg gtggtagttt tcttgtttta 660
ttgccactag tctctagtca gtgtgttaat cttacataac aagctt 706
Claims (10)
1. A SARS-ConV-2 virus S protein extramembranous BD end domain high targeting recombinant protein is characterized in that the nucleotide sequence of the encoded recombinant protein is shown as SEQ ID NO: 5 or as shown in SEQ ID NO: and 6.
2. A nucleic acid molecule having a nucleotide sequence set forth in SEQ ID NO: 5 or SEQ ID NO: and 6.
3. A recombinant expression vector capable of expressing the SARS-ConV-2 virus S protein extramembranous BD-terminal domain highly targeted recombinant protein of claim 1, wherein the recombinant expression vector comprises the nucleic acid molecule of claim 2.
4. The recombinant expression vector of claim 3, wherein the recombinant expression vector comprises one of an expression plasmid vector, a phage vector, and a viral vector.
5. The recombinant expression vector according to claim 4, wherein the nucleotide sequence of the expression region of the expression plasmid vector is as set forth in SEQ ID NO: 5 or as shown in SEQ ID NO: and 6.
6. The recombinant expression vector of claim 5, wherein the expression plasmid vectors are pET28a-DTT-2Sc and pET28a-DTT-3 Se.
7. An engineered bacterium or cell comprising the recombinant expression vector of any one of claims 3-6.
8. A preparation method of BD end domain high targeting recombinant protein outside a SARS-ConV-2 virus S protein membrane is characterized in that the method comprises the following steps:
obtaining antigenic determinants Sc and Se of the BD end structural domain outside the SARS-ConV-2 virus S protein membrane, wherein the nucleotide sequence of the Sc is shown as SEQ ID NO: 1, and the nucleotide sequence of Se is shown as SEQ ID NO: 2 is shown in the specification;
constructing and obtaining the Sc and Se expression plasmid, wherein the nucleotide sequence of the Sc expression plasmid expression region is shown as SEQ ID NO: 5, the nucleotide sequence of the expression region of the Se expression plasmid is shown as SEQ ID NO: 6 is shown in the specification;
and respectively carrying out secretory expression and purification on the Sc and Se expression plasmids to obtain the purified recombinant SARS-ConV-2 virus S protein extramembranous BD end domain high-targeting recombinant protein.
9. A novel coronavirus subunit vaccine comprising the recombinant protein of claim 1 and a pharmaceutically acceptable adjuvant.
10. The novel coronavirus subunit vaccine of claim 9, wherein the adjuvant comprises at least one of aluminium hydroxide, lecithin, freund's adjuvant, MPL TM, IL-12, aluminium hydroxide in combination with CpG ODN complex adjuvant, ISA51VG, ISA720VG, MF59, QS21, AS03 adjuvant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110411059.XA CN113151331B (en) | 2021-04-16 | 2021-04-16 | SARS-ConV-2 virus S protein extramembranous BD end domain high targeting recombinant protein and subunit vaccine thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110411059.XA CN113151331B (en) | 2021-04-16 | 2021-04-16 | SARS-ConV-2 virus S protein extramembranous BD end domain high targeting recombinant protein and subunit vaccine thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113151331A true CN113151331A (en) | 2021-07-23 |
| CN113151331B CN113151331B (en) | 2022-08-30 |
Family
ID=76868639
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110411059.XA Active CN113151331B (en) | 2021-04-16 | 2021-04-16 | SARS-ConV-2 virus S protein extramembranous BD end domain high targeting recombinant protein and subunit vaccine thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113151331B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112979766A (en) * | 2021-04-16 | 2021-06-18 | 武汉大学 | SARS-ConV-2 virus S protein membrane outside N end structure domain high targeting recombinant protein and subunit vaccine thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060240515A1 (en) * | 2003-07-21 | 2006-10-26 | Dimitrov Dimiter S | Soluble fragments of the SARS-CoV spike glycoprotein |
| CN111217919A (en) * | 2020-03-04 | 2020-06-02 | 中山大学 | Novel coronavirus S protein double-region subunit nano vaccine based on pyrococcus ferritin |
| CN111217918A (en) * | 2020-03-04 | 2020-06-02 | 中山大学 | Novel coronavirus S protein double-region subunit nano vaccine based on 2, 4-dioxotetrahydropteridine synthase |
| CN112043825A (en) * | 2020-07-13 | 2020-12-08 | 中国医学科学院医学生物学研究所 | Subunit vaccine for preventing novel coronavirus infection based on novel coronavirus spike protein S1 region |
| CN112076315A (en) * | 2020-08-25 | 2020-12-15 | 中国农业科学院生物技术研究所 | Nano antigen particle fused with new coronavirus S protein and ferritin subunit, new coronavirus vaccine, and preparation method and application thereof |
| CN112094326A (en) * | 2020-09-25 | 2020-12-18 | 河南省生物工程技术研究中心 | Novel coronavirus antigen and application thereof |
| WO2020257317A1 (en) * | 2019-06-18 | 2020-12-24 | Citranvi Biosciences, Llc | Rationally engineered carrier proteins for vaccines |
| CN112500498A (en) * | 2020-02-26 | 2021-03-16 | 四川大学 | Novel coronavirus vaccine and preparation method and application thereof |
-
2021
- 2021-04-16 CN CN202110411059.XA patent/CN113151331B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060240515A1 (en) * | 2003-07-21 | 2006-10-26 | Dimitrov Dimiter S | Soluble fragments of the SARS-CoV spike glycoprotein |
| WO2020257317A1 (en) * | 2019-06-18 | 2020-12-24 | Citranvi Biosciences, Llc | Rationally engineered carrier proteins for vaccines |
| CN112500498A (en) * | 2020-02-26 | 2021-03-16 | 四川大学 | Novel coronavirus vaccine and preparation method and application thereof |
| CN111217919A (en) * | 2020-03-04 | 2020-06-02 | 中山大学 | Novel coronavirus S protein double-region subunit nano vaccine based on pyrococcus ferritin |
| CN111217918A (en) * | 2020-03-04 | 2020-06-02 | 中山大学 | Novel coronavirus S protein double-region subunit nano vaccine based on 2, 4-dioxotetrahydropteridine synthase |
| CN112043825A (en) * | 2020-07-13 | 2020-12-08 | 中国医学科学院医学生物学研究所 | Subunit vaccine for preventing novel coronavirus infection based on novel coronavirus spike protein S1 region |
| CN112076315A (en) * | 2020-08-25 | 2020-12-15 | 中国农业科学院生物技术研究所 | Nano antigen particle fused with new coronavirus S protein and ferritin subunit, new coronavirus vaccine, and preparation method and application thereof |
| CN112094326A (en) * | 2020-09-25 | 2020-12-18 | 河南省生物工程技术研究中心 | Novel coronavirus antigen and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| HASANAIN ABDULHAMEED ODHAR等: "Towards the design of epitope candidates for Coronavirus 2", 《BIOMEDICAL INFORMATICS》 * |
| 杨海龙等: "新冠病毒刺突蛋白及其受体的结构与相互作用", 《大学化学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112979766A (en) * | 2021-04-16 | 2021-06-18 | 武汉大学 | SARS-ConV-2 virus S protein membrane outside N end structure domain high targeting recombinant protein and subunit vaccine thereof |
| CN112979766B (en) * | 2021-04-16 | 2022-08-16 | 武汉大学 | SARS-ConV-2 virus S protein membrane outside N end structure domain high targeting recombinant protein and subunit vaccine thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113151331B (en) | 2022-08-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111592602B (en) | Beta coronavirus antigen, preparation method and application thereof | |
| CN111217919A (en) | Novel coronavirus S protein double-region subunit nano vaccine based on pyrococcus ferritin | |
| ES2659875T3 (en) | Biochemically stabilized HIV-1 Env trimer vaccine | |
| CN111662389A (en) | SARS-CoV-2 fusion protein and vaccine composition thereof | |
| CN112386684B (en) | COVID-19 vaccine and preparation method and application thereof | |
| WO2021147025A1 (en) | Anti 2019-ncov vaccine | |
| CN112094327A (en) | Truncation body based on novel coronavirus RBD-SD1 protein and application thereof | |
| CN113354717A (en) | Novel coronavirus SARS-CoV-2 broad-spectrum polypeptide antigen and its specific neutralizing antibody and application | |
| WO2023217206A1 (en) | Novel coronavirus chimeric nucleic acid vaccine and use thereof | |
| US20120100165A1 (en) | Drug transporter, and adjuvant and vaccine each utilizing same | |
| CN113151184A (en) | Method for cell membrane-based display of coronavirus immunogens to induce neutralizing antibodies | |
| CN114437185A (en) | Coronavirus trimer subunit vaccine and application thereof | |
| Shao et al. | An engineered biomimetic MPER peptide vaccine induces weakly HIV neutralizing antibodies in mice | |
| CN103864936B (en) | HPV18 type L2NE7E6 antigen-4 fusion protein genes, expression vector, method, bacterial strain and purposes | |
| CN113151331B (en) | SARS-ConV-2 virus S protein extramembranous BD end domain high targeting recombinant protein and subunit vaccine thereof | |
| WO2022166806A1 (en) | Novel cd271-based antigenic epitope and application thereof | |
| EP3004173A1 (en) | Single domain antibody display | |
| WO2024002129A1 (en) | Novel coronavirus trimer chimeric vaccine and use thereof | |
| CN112979766B (en) | SARS-ConV-2 virus S protein membrane outside N end structure domain high targeting recombinant protein and subunit vaccine thereof | |
| CN116693632A (en) | African swine fever virus epitope peptide, monoclonal antibody and application thereof | |
| CN105777909A (en) | A-type foot-and-mouth disease targeting composite epitope protein mediated by pig chemotactic factors and vaccine | |
| CN114057850B (en) | Polypeptide and immunogenic conjugate for preventing novel coronavirus COVID-19 and application thereof | |
| Majidi et al. | Expression and Purification of Brucella spp. Lumazine Synthase Decameric Carrier in Fusion to Extracellular Domain of Influenza M2E Protein | |
| WO2022106860A1 (en) | Recombinant peptides for use in therapy | |
| CN112316130A (en) | SARS-CoV2 mucosal immune vaccine and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |