CN117448354A - Recombinant DNA molecule for encoding coronavirus antigen, DNA vaccine and application - Google Patents
Recombinant DNA molecule for encoding coronavirus antigen, DNA vaccine and application Download PDFInfo
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- CN117448354A CN117448354A CN202210874767.1A CN202210874767A CN117448354A CN 117448354 A CN117448354 A CN 117448354A CN 202210874767 A CN202210874767 A CN 202210874767A CN 117448354 A CN117448354 A CN 117448354A
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- A61P31/14—Antivirals for RNA viruses
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Abstract
The invention relates to the field of biotechnology, in particular to a recombinant DNA molecule for encoding coronavirus antigen, a DNA vaccine and application thereof. The invention recombines the nucleic acid molecule encoding the S protein RBD region of the novel coronal SARS-CoV-2Beta mutant strain with the nucleic acid molecule encoding the S protein RBD region of the novel coronal SARS-CoV-2Omicron mutant strain, the polypeptide encoded by the recombined nucleic acid molecule has the double immunogenicity of the S protein RBD region of the novel coronal SARS-CoV-2Beta mutant strain and the S protein RBD region of the novel coronal SARS-CoV-2Omicron mutant strain, and when the polypeptide with double immunogenicity is used as a vaccine immune effect component, the induced neutralizing antibody can simultaneously form immune reaction aiming at the S protein RBD region of the novel coronal SARS-CoV-2Beta mutant strain and the S protein RBD region of the novel coronal SARS-CoV-2Omicron mutant strain, thereby being capable of inducing specific humoral immunity and cellular immune response.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a DNA molecule for encoding coronavirus antigen, a DNA vaccine and application thereof.
Background
Coronaviruses belong to the family Coronaviridae (Coronaviridae), including four genera of alpha-coronavirus, beta-coronavirus, gamma-coronavirus and delta-coronavirus, the novel coronavirus currently prevalent (Severe acute respiratory syndrome coronavirus, SARS-CoV-2) belongs to the coronavirus of the beta genus, and is mainly transmitted through respiratory droplets, and pneumonia (Novel Coronavirus-infected Pneumonia, NCP) can also be caused by contact transmission, so that the population is generally susceptible.
Currently, the world health organization is classified into two main categories in the classification of new coronavirus mutants, one category being a worrying mutant strain (VOC) and the other category being a mutant strain (VOI) of interest. Up to now there are 5 VOC mutants, alpha (Alpha, accession number b.1.1.7), beta (Beta, accession number b.1.351), gamma (Gamma, accession number p.1), delta (Delta, accession number b.1.617.2) and omicker (Omicron, accession number b.1.1.529) variants, respectively; the 8 VOI mutants were, respectively, epothilone (ep silon, accession No. b.1.427/429), zeta (Zeta, accession No. p.2), sita (Theta, accession No. P.3), eta (Eta, accession No. b.1.525), about tower (Iota, accession No. b.1.526), kappa (Kappa, accession No. b.1.617.1), lambda (Lambda, accession No. c.37), and muu (Mu, accession No. b.1.621). In particular, recent outbreaks of the omucon variant have 15 mutation sites in the Receptor Binding Domain (RBD) due to more than 30 mutations in the Spike segment of the major protective antigen, 11 of which are in the Receptor Binding Motif (RBM) region, including three mutations that were previously present in beta and gamma mutants: 417 484, and 501, wherein N501Y is also present in the alpha mutant. Most of the RBD antibodies bind to RBM regions, and when the RBM regions are mutated, the antibodies bound to these regions are easily affected and escape. It is worth mentioning that another 4 mutation sites not in the RBM region are distributed at the interaction interface of S monomers, which may affect the stability of trimer. In addition, of the 15 mutations, 9 sites were not conserved between SARS-CoV-2S and SARS-CoV S, and the mutation occurred in the non-conserved region. While the mutation sites of the amino-terminal domain (NTD) are concentrated around: A67V/Del69-70; T95I; G142D/Del143-145; del211/L212I, which is structurally concentrated on the NTD surface and is also a common recognition site for novel crown NTD antibodies; thus, simultaneous mutation of these sites also affects the affinity and neutralizing activity of the NTD antibodies. Based on the existing knowledge of mutation sites, the variant breaks through the protection of most of neutralizing antibodies, and part of previous data shows that the variant reduces the neutralizing antibodies of the novel crown vaccine by about 40 times compared with the wild type, and based on various frequent variants and the appearance of super transmissible Omikovia, the development of a broad-spectrum vaccine which is 'in a non-allergic manner' is an effective means for coping with the frequent variants.
In view of this, the present invention has been made.
Disclosure of Invention
It is an object of the present invention to provide a recombinant nucleic acid molecule encoding SARS-CoV-2 virus antigen, which is expected to achieve a broad-spectrum immune effect "in the absence of an allergic strain".
It is a second object of the present invention to provide a biological material comprising the recombinant nucleic acid molecule described above.
It is a further object of the present invention to provide the use of the above biological material.
It is a fourth object of the present invention to provide a DNA vaccine of SARS-CoV-2 virus comprising the recombinant nucleic acid molecule and/or biological material described above.
The fifth object of the present invention is to provide a method for producing the DNA vaccine.
In order to solve the technical problems and achieve the purposes, the invention provides the following technical scheme:
in a first aspect, the present invention provides a recombinant nucleic acid molecule comprising any one of (a) to (c) as follows:
(a) The recombinant DNA molecules comprise a first DNA molecule encoding the S protein RBD region of the novel SARS-CoV-2Beta mutant strain and a second DNA molecule encoding the S protein RBD region of the novel SARS-CoV-2 Omicron mutant strain;
(b) A DNA molecule derived from (a) by substitution, deletion or addition of one or several nucleotides in the nucleotide sequence of the recombinant DNA molecule defined in (a) and having the function of encoding the S protein RBD region of the novel SARS-CoV-2Beta mutant strain and encoding the S protein RBD region of the novel SARS-CoV-2 Omicron mutant strain;
(c) Nucleic acid molecules which hybridize under stringent conditions with the nucleotide sequence of the recombinant DNA molecule defined in (a) or the DNA molecule defined in (b) and which encode the S protein RBD region of the novel SARS-CoV-2Beta mutant strain and the S protein RBD region of the novel SARS-CoV-2 Omicron mutant strain.
In an alternative embodiment, the amino acid sequence encoded by the first DNA molecule is shown in SEQ ID No. 1; the amino acid sequence of the second DNA molecule is shown as SEQ ID No. 2.
In an alternative embodiment, the nucleotide sequence of the first nucleic acid molecule is set forth in SEQ ID No. 3; the nucleotide sequence of the second nucleic acid molecule is shown as SEQ ID No. 4.
Preferably, the nucleotide sequence of the recombinant nucleic acid molecule is shown as SEQ ID No.5 or has at least 90% identity with the nucleotide sequence shown as SEQ ID No.5, and encodes the RBD region of the S protein of the novel SARS-CoV-2Beta mutant strain and the RBD region of the S protein of the novel SARS-CoV-2 Omicron mutant strain.
In a second aspect, the present invention provides a biomaterial comprising:
a construct comprising a recombinant nucleic acid molecule according to any one of the preceding embodiments; and, a third nucleic acid molecule encoding a signal peptide linked to the 5' end of the recombinant nucleic acid molecule;
(ii) recombinant expression vectors, including original expression vectors; and, inserting into the original expression vector a coding nucleic acid fragment selected from the recombinant nucleic acid molecule of any one of the preceding embodiments or the (i) construct; preferably, the original expression vector is a pVAX1 plasmid;
(iii) a transformant obtained by introducing the recombinant expression vector of (ii) into a host cell selected from the group consisting of insect cells, yeast, avian cells, and mammalian cells; preferably, the host cell is HEK293, CHO or COS-7;
(iv) a polypeptide comprising a polypeptide encoded by the recombinant nucleic acid molecule of any one of the preceding embodiments, a polypeptide encoded by the construct of (i), or a polypeptide obtained by expression of a transformant of (iii);
(V) an antibody that specifically binds to the (IV) polypeptide.
In an alternative embodiment, the signal peptide encoded by the third nucleic acid molecule comprises the following (c) or (d):
(c) A signal peptide with an amino acid sequence shown as SEQ ID No. 6;
(d) A signal peptide derived from (c) having a signal peptide function by substituting, deleting or adding one or more amino acids in the amino acid sequence of the signal peptide defined in (c).
In a third aspect, the invention provides the use of a recombinant nucleic acid molecule according to any one of the preceding embodiments or a biological material according to any one of the preceding embodiments in (a) or (B) as follows:
(A) Preparing vaccine for preventing and/or treating SARS-CoV-2 virus infection;
(B) Preparing medicine for preventing and/or treating SARS-CoV-2 virus related diseases.
In alternative embodiments, the SARS-CoV-2 virus comprises a wild-type strain, a B.1.617.2 mutant, a B.1.1.7 mutant, a B.1.351 mutant, a P.1 mutant, a B.1.2 mutant, a B.1 mutant, a B.1.621 mutant, a B.1.525 mutant, a B.1.526 mutant, a C.37 mutant, a B.1.617.1 mutant or a B.1.1.529 mutant.
In a fourth aspect, the invention provides a DNA vaccine comprising the recombinant nucleic acid molecule of any one of the preceding embodiments or the recombinant expression vector of any one of the preceding embodiments.
In alternative embodiments, the DNA vaccine further comprises at least one of a pharmaceutically acceptable adjuvant, carrier, diluent, or excipient;
and/or at least one drug having a therapeutic effect on SARS-CoV-2 virus;
preferably, the adjuvant comprises at least one of an aluminium adjuvant, TLRs ligand, metal ion, cytokine or chemokine adjuvant;
further preferably, the metal ion comprises Mn 2+ And/or Zn 2+ 。
In an alternative embodiment, the recombinant nucleic acid molecule of any one of the preceding embodiments or the recombinant expression vector of any one of the preceding embodiments is introduced into a host cell and cultured, and the recombinant nucleic acid molecule or the recombinant expression vector in the host cell is extracted to obtain a DNA vaccine.
In a fifth aspect, the present invention provides the use of a DNA vaccine according to the preceding embodiments in (i) to (iii) as follows:
(i) Regulating immunity of organism;
(ii) anti-SARS-CoV-2 virus infection;
(iii) Prevent immunopathogenic injury.
Preferably, the SARS-CoV-2 virus comprises a wild-type strain, a B.1.617.2 mutant, a B.1.1.7 mutant, a B.1.351 mutant, a P.1 mutant, a B.1.2 mutant, a B.1 mutant, a B.1.621 mutant, a B.1.525 mutant, a B.1.526 mutant, a C.37 mutant, a B.1.617.1 mutant or a B.1.1.529 mutant.
In a sixth aspect, the present invention provides a method for preventing and/or treating infection of a mammal with SARS-CoV-2 virus, said method comprising vaccinating said mammal with the DNA vaccine described above.
Preferably, the SARS-CoV-2 virus comprises a wild-type strain, a B.1.617.2 mutant, a B.1.1.7 mutant, a B.1.351 mutant, a P.1 mutant, a B.1.2 mutant, a B.1 mutant, a B.1.621 mutant, a B.1.525 mutant, a B.1.526 mutant, a C.37 mutant, a B.1.617.1 mutant or a B.1.1.529 mutant.
Preferably, the mammal is a human.
The invention recombines the nucleic acid molecule encoding the S protein RBD region of the novel coronal SARS-CoV-2Beta mutant strain with the nucleic acid molecule encoding the S protein RBD region of the novel coronal SARS-CoV-2 Omicron mutant strain, the polypeptide encoded by the recombined nucleic acid molecule has the double immunogenicity of the S protein RBD region of the novel coronal SARS-CoV-2Beta mutant strain and the S protein RBD region of the novel coronal SARS-CoV-2 Omicron mutant strain, and when the polypeptide with double immunogenicity is used as a vaccine immune effect component, the synthesized neutralizing antibody can simultaneously form immune reaction against the S protein RBD region of the novel coronal SARS-CoV-2Beta mutant strain and the S protein RBD region of the novel coronal SARS-CoV-2 Omicron mutant strain. Meanwhile, the double antigen design of SARS-CoV-2 mutant strain is adopted to amplify the immune effect of the conservative epitope in SARS-CoV-2 relativity as far as possible, thus avoiding the antigen drift caused by continuous mutation in SARS-CoV-2 relativity and realizing the broad-spectrum immune effect of 'in non-allergic ten thousand strain'. Meanwhile, compared with the serial sequence of the Omicron-Beta RBD, the serial sequence of the Beta-Omicron RBD selected on the protein domain serial strategy ensures that Beta-folding of two antigen domains of the Beta RBD and the Omicron RBD on the space structure is more regular, and the more regular protein space structure can lead the antigen-antibody reaction to be better and the immune effect to be better.
Based on the beneficial effects of the recombinant nucleic acid molecules, the invention also provides a preparation method of the recombinant nucleic acid molecules, biological materials used for preparing the recombinant nucleic acid molecules, polypeptides obtained by expressing the recombinant nucleic acid molecules, and DNA vaccine taking the biological materials as main immune components and a preparation method of the DNA vaccine, which are verified to be capable of effectively transcribing and expressing in mammalian cells and also have good immunogenicity, and can obviously excite experimental animals to generate antigen-specific antibodies on the 14 th day after primary immunization and the 7 th day after boosting immunization and on the 14 th day for humoral immune response, so that antibodies against new crown wild type antigens, new crown Delta mutant antigens, beta mutant antigens and Omicron mutant antigens can be generated, and the DNA vaccine has neutralization activity; for cellular immune responses, the DNA vaccine is capable of inducing not only high levels of antigen-specific IFN- γ responses, but also the production of antigen-specific CD4 ifnγ and CD4 TNF- α T cell subsets against the novel crown wild-type antigen, novel crown Delta mutant antigen, and omacron mutant antigen; the humoral immunity and the cell immunity experiments show that the DNA vaccine has good immunogenicity and broad spectrum.
Based on this, the DNA vaccine provided by the present invention can regulate the immune function of organism, effectively prevent SARS-CoV-2 virus and its mutant strain infection, and simultaneously can intervene in the treatment of diseases caused by SARS-CoV-2 virus and its mutant strain.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the result of scoring the codon optimization index of the DNA sequence provided in example 1 of the present invention;
FIG. 2 is a graph showing GC content scoring results after DNA sequence optimization provided in example 1 of the present invention;
FIG. 3 is a graph showing the result of scoring the number of negative regulatory elements after optimizing the DNA sequence provided in example 1 of the present invention;
FIG. 4 is a graph showing the result of qPCR expression fold after optimizing the DNA sequence provided in example 1 of the present invention;
FIG. 5 is a structural analysis of the binding protein according to example 1 of the present invention;
FIG. 6 shows qPCR expression results of a novel coronavirus broad-spectrum candidate DNA vaccine provided in example 3 of the present invention;
FIG. 7 shows the result of Western Blot detection of antigen protein of a novel coronal wild strain and coronavirus broad-spectrum candidate DNA vaccine provided in example 4 of the present invention;
FIG. 8 shows the result of detecting antigen protein by ELISA method of broad-spectrum candidate DNA vaccine of coronavirus of novel coronal wild strain provided in example 5 of the present invention;
FIG. 9 shows the results of antigen-specific antibodies on day 14 after primary immunization of a novel coronavirus broad-spectrum candidate DNA vaccine provided in example 6 of the present invention;
FIG. 10 shows the results of antigen-specific antibodies on day 14 after booster immunization with the novel coronavirus broad-spectrum candidate DNA vaccine provided in example 6 of the present invention;
FIG. 11 shows the neutralizing antibody results of the novel coronavirus broad-spectrum candidate DNA vaccine provided in example 6 of the present invention on day 7 after booster immunization;
FIG. 12 is a comparison of neutralizing antibodies at day 14 after booster immunization for each vaccine provided in example 6 of the present invention;
FIG. 13 shows the results of antigen-specific IFN-. Gamma.ELISOPT on day 14 after primary immunization of a novel coronavirus broad-spectrum candidate DNA vaccine provided in example 6 of the present invention;
FIG. 14 shows the results of antigen-specific IFN-. Gamma.ELISOPT on day 14 after booster immunization with the novel coronavirus broad-spectrum candidate DNA vaccine provided in example 6 of the present invention;
FIG. 15 shows the results of antigen-specific CD4 IFN-gamma T cell subsets of the novel coronavirus broad-spectrum candidate DNA vaccine provided in example 6 of the present invention on day 14 after primary immunization;
FIG. 16 shows the results of antigen-specific CD4 TNF-. Alpha.T cell subset on day 14 after primary immunization of a novel coronal wild type strain, coronal virus broad-spectrum candidate DNA vaccine provided in example 6 of the present invention;
FIG. 17 shows the results of antigen-specific CD4 IFN-gamma T cell subsets on day 14 after booster immunization with the novel coronal wild strain, coronavirus broad-spectrum candidate DNA vaccine provided in example 6 of the present invention;
FIG. 18 shows the results of antigen-specific CD4 TNF-. Alpha.T cell subset on day 14 after booster immunization with the novel coronal wild strain, coronavirus broad-spectrum candidate DNA vaccine provided in example 6 of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally well known in the art and are performed according to conventional methods as described in various general and more specific references cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly accomplished in the art, or as described herein. Nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques therefor, are those well known and commonly employed in the art.
Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that: like reference numerals and letters denote like items in the following figures, and thus once an item is defined in one figure, no further definition or explanation thereof is necessary in the following figures. Furthermore, the terms "first," "second," "third," and the like are used merely to distinguish between descriptions and should not be construed as indicating or implying relative importance.
In a first embodiment, the present invention provides a recombinant nucleic acid molecule comprising any one of the following (a) to (c):
(a) The recombinant DNA molecules comprise a first DNA molecule encoding the S protein RBD region of the novel SARS-CoV-2Beta mutant strain and a second DNA molecule encoding the S protein RBD region of the novel SARS-CoV-2Omicron mutant strain;
(b) A DNA molecule derived from (a) having the functions of a novel SARS-CoV-2Beta mutant strain S protein RBD region and a novel SARS-CoV-2Omicron mutant strain S protein RBD region encoded by substitution, deletion or addition of one or several nucleotides in the nucleotide sequence of the recombinant DNA molecule defined in (a);
(c) Nucleic acid molecules which hybridize under stringent conditions with the nucleotide sequence of the recombinant DNA molecule defined in (a) or the DNA molecule defined in (b) and which encode the S protein RBD region of the novel SARS-CoV-2Beta mutant strain and the S protein RBD region of the novel SARS-CoV-2Omicron mutant strain.
It should be noted that the present invention is not limited to the above-described sequence of the first DNA molecule and the second DNA molecule, and in some embodiments the recombinant nucleic acid molecule comprises the first DNA molecule and the second DNA molecule sequentially connected from the 5 'end to the 3' end, and in other embodiments the recombinant nucleic acid molecule obtained by exchanging the sequence of the first DNA molecule and the second DNA molecule may still have a broad spectrum of immunogenicity. Those skilled in the art can choose the connection sequence of the first DNA molecule and the second DNA molecule according to specific experimental conditions and actual requirements, and the connection manner given in the embodiment of the present invention is only an example, and is not limited to the connection sequence.
It will be understood that the above-mentioned recombinant nucleic acid molecule (c) hybridizes with the recombinant DNA molecule (a) or the DNA molecule (b) under "stringent conditions" means a recombinant nucleic acid molecule (c) obtained by hybridization using the recombinant DNA molecule (a) or the DNA molecule (b) as a template in accordance with the base complementary pairing rules. Also, it will be appreciated by those skilled in the art that the recombinant nucleic acid molecule (c) includes both the DNA molecules obtained by replication and the various RNA molecules obtained by transcription.
In addition, the nucleotide sequence provided by the invention is obtained by optimizing an unique codon optimization system.
In an alternative embodiment, the amino acid sequence encoded by the first DNA molecule is shown in SEQ ID No. 1; the amino acid sequence of the second DNA molecule is shown as SEQ ID No. 2.
It should be noted that, the S protein RBD region fragment of the novel SARS-CoV-2Beta mutant strain and the S protein RBD region fragment of the novel SARS-CoV-2 Omicron mutant strain have mutual influence in the process of folding polypeptide space conformation, and the Beta-folding of two antigen domains of the Beta RBD and the Omicron RBD is more regular due to the more regular protein space structure compared with the serial sequence of the Omicron-Beta RBD, so that the antigen-antibody reaction is better due to the more regular protein space structure, and the immune effect is better. The SEQ ID No.1 and the SEQ ID No.2 are optimal combined sequences which have complete functions and small mutual interference of the selected novel SARS-CoV-2Beta mutant strain S protein RBD region and the novel SARS-CoV-2 Omicron mutant strain S protein RBD region, aiming at realizing wide-spectrum long-acting immunogenicity of 'non-allergic ten-thousand'. From the foregoing, it will be appreciated that there are a variety of additional novel S protein RBD regions of the SARS-CoV-2Beta mutant strain that can be used in the encoding of the first DNA molecule of the present invention, e.g., derived polypeptides from SEQ ID No.1, which have been deleted or added at the 5 'and/or 3' end by one or more amino acids. Similarly, the novel SARS-CoV-2 Omicron mutant S protein RBD region encoded by the second DNA molecule of the present invention also comprises derivative polypeptide obtained by deleting or adding one or more amino acids at the 5 'end and/or 3' end of SEQ ID No. 1.
In an alternative embodiment, the nucleotide sequence of the first nucleic acid molecule is set forth in SEQ ID No. 3; the nucleotide sequence of the second nucleic acid molecule is shown as SEQ ID No. 4.
Preferably, the nucleotide sequence of the recombinant nucleic acid molecule is shown as SEQ ID No.5 or has at least 90% identity with the nucleotide sequence shown as SEQ ID No.5, and encodes the RBD region of the S protein of the novel SARS-CoV-2Beta mutant strain and the RBD region of the S protein of the novel SARS-CoV-2 Omicron mutant strain.
It is understood that in the present invention, "identity" refers to similarity between nucleotide sequences, including nucleotide sequences having at least 90% (e.g., may be, but not limited to, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to the nucleotide sequence set forth in SEQ ID No.5 of the present invention.
In a second aspect, the present invention provides a biomaterial comprising:
a construct comprising a recombinant nucleic acid molecule according to any one of the preceding embodiments; and, a third nucleic acid molecule encoding a signal peptide linked to the 5' end of the recombinant nucleic acid molecule;
(ii) recombinant expression vectors, including original expression vectors; and, inserting into the original expression vector a coding nucleic acid fragment selected from the recombinant nucleic acid molecule of any one of the preceding embodiments or the (i) construct; preferably, the original expression vector is a pVAX1 plasmid;
(iii) a transformant obtained by introducing the recombinant expression vector of (ii) into a host cell selected from the group consisting of insect cells, yeast, avian cells, and mammalian cells; preferably, the host cell is HEK293, CHO or COS-7;
(iv) a polypeptide comprising a polypeptide encoded by the recombinant nucleic acid molecule of any one of the preceding embodiments, a polypeptide encoded by the construct of (i), or a polypeptide obtained by expression of a transformant of (iii);
(V) antibodies, such as monoclonal or polyclonal antibodies, that specifically bind to the (IV) polypeptide.
It should be noted that the original expression vector may be a eukaryotic expression vector, and the protein encoded by the DNA molecule is produced by cellular transcription and translation mechanisms. Alternatively, the original expression vector may have expression signals such as a strong promoter, a strong stop codon, regulation of the distance between the promoter and the cloned gene, and transcription termination sequence insertion. Preferably, the eukaryotic expression vector includes pVAX1, but is not limited to any other expression vector capable of expressing DNA and enabling cells to translate sequences into antigens recognized by the immune system.
For the above-mentioned host cells, which are only typical preferred cells according to the present invention, those skilled in the art can select other suitable host cells according to the actual experimental conditions or actual requirements, and are not limited to eukaryotic cells or prokaryotic cells.
It can be appreciated that the biomaterial provided by the invention can be directly applied to production of different requirements and scenes as a biological module.
In an alternative embodiment, the signal peptide encoded by the third nucleic acid molecule comprises the following (c) or (d):
(c) A signal peptide with an amino acid sequence shown as SEQ ID No. 6;
(d) A signal peptide derived from (c) having a signal peptide function by substituting, deleting or adding one or more amino acids in the amino acid sequence of the signal peptide defined in (c).
The signal peptide is matched with the efficient expression of SARS-CoV-2 virus gene, and can raise the expression efficiency of the recombinant nucleic acid molecule in host obviously.
In a third aspect, the invention provides the use of a recombinant nucleic acid molecule according to any one of the preceding embodiments or a biological material according to any one of the preceding embodiments in (a) or (B) as follows:
(A) Preparing vaccine for preventing and/or treating SARS-CoV-2 virus infection;
(B) Preparing medicine for preventing and/or treating related diseases caused by SARS-CoV-2 virus, including lung injury, brain injury, liver and kidney injury or heart injury.
In alternative embodiments, the SARS-CoV-2 virus comprises a wild-type strain, a B.1.617.2 mutant, a B.1.1.7 mutant, a B.1.351 mutant, a P.1 mutant, a B.1.2 mutant, a B.1 mutant, a B.1.621 mutant, a B.1.525 mutant, a B.1.526 mutant, a C.37 mutant, a B.1.617.1 mutant or a B.1.1.529 mutant.
In a fourth aspect, the invention provides a DNA vaccine comprising the recombinant nucleic acid molecule of any one of the preceding embodiments or the recombinant expression vector of any one of the preceding embodiments.
The DNA vaccine not only can be effectively transcribed and expressed in mammalian cells, but also has good immunogenicity, and can obviously excite experimental animals to generate antigen-specific antibodies on the 14 th day after primary immunization and the 7 th day after booster immunization and the 14 th day for humoral immune response, so that antibodies aiming at new crown wild type antigens can be generated, and antibodies aiming at new crown Delta mutant antigens, beta mutant antigens and Omicron mutant antigens can be generated, and the DNA vaccine has neutralization activity; for cellular immune responses, the DNA vaccine is capable of inducing not only high levels of antigen-specific IFN- γ responses, but also the production of antigen-specific CD4 ifnγ and CD4 TNF- α T cell subsets against the novel crown wild-type antigen, novel crown Delta mutant antigen, and omacron mutant antigen; the humoral immunity and the cell immunity experiments show that the DNA vaccine has good immunogenicity and broad spectrum.
In some embodiments, the DNA vaccine further comprises at least one of a pharmaceutically acceptable adjuvant, carrier, diluent, or excipient to increase the ability of its active ingredient DNA molecule to generate an immune response in a subject. The adjuvant comprises at least one of aluminum adjuvant, TLRs ligand, metal ion, cytokine or chemokine adjuvant; preferably, the metal ions include Mn 2+ And/or Zn 2+ 。
In other embodiments, the DNA vaccine further comprises at least one drug having a therapeutic effect on SARS-CoV-2 virus to enhance the therapeutic effect of the vaccine on a disease associated with SARS-COV-2 virus.
In still other embodiments, the DNA vaccine comprises at least one of the pharmaceutically acceptable adjuvants, carriers, diluents or excipients described above and at least one drug having therapeutic effect on SARS-CoV-2 virus.
In an alternative embodiment, a recombinant nucleic acid molecule according to any one of the preceding embodiments or a recombinant expression vector according to any one of the preceding embodiments is introduced into a host cell and cultured, and the recombinant nucleic acid molecule or recombinant expression vector in the host cell is extracted to prepare a DNA vaccine.
In a fifth aspect, the present invention provides a method for preventing and/or treating infection of a mammal with SARS-CoV-2 virus, said method comprising vaccinating said mammal with the DNA vaccine described above.
In alternative embodiments, the SARS-CoV-2 virus comprises a wild-type strain, a B.1.617.2 mutant, a B.1.1.7 mutant, a B.1.351 mutant, a P.1 mutant, a B.1.2 mutant, a B.1 mutant, a B.1.621 mutant, a B.1.525 mutant, a B.1.526 mutant, a C.37 mutant, a B.1.617.1 mutant or a B.1.1.529 mutant.
In an alternative embodiment, the mammal is a human.
The DNA vaccine provided by the invention has the following action mechanism: the optimized nucleotide sequence of the S protein RBD region of the mutant strain S protein encoding the novel SARS-CoV-2Beta is connected in series with the optimized nucleotide sequence of the S protein RBD region of the mutant strain S protein encoding the novel SARS-CoV-2 Omicron, and after the efficient expression signal peptide is added, the eukaryotic expression vector is inserted into the host cell, so that the eukaryotic expression vector efficiently expresses virus antigen in the host cell, and the antiviral humoral immune response and the cellular immune response are systematically activated through the antigen presenting process. Antibodies raised by the activated humoral immune response may prevent viral entry, and the activated cellular immune response may further clear virus-infected cells and modulate adverse reactions due to potential side effects of ADE.
Based on the action mechanism, the invention also provides application of the DNA vaccine, which comprises the following steps:
(i) Regulating immunity of organism;
(ii) anti-SARS-COV-2 virus infection;
(iii) Prevent immunopathogenic injury.
Some embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following embodiments and features of the embodiments may be combined with each other without conflict.
Example 1: optimized screening of nucleic acid encoding S protein RBD region protein
In order to increase the protein expression of a target protein in a host cell, the nucleic acid sequence of the target gene needs to be optimized, and the general principle of nucleic acid sequence optimization is as follows: (1) Optimizing degenerate codons according to the preference of the host cell for nucleic acid codons, so that the optimized sequence contains more nucleic acid codons which are favorable for the host cell to recognize; (2) Further optimizing the GC content in the nucleic acid sequence on the basis of codon preference optimization, so that the sequence with optimized GC content can express more target proteins; (3) Optimizing the nucleic acid sequence to enable the nucleic acid sequence to transcribe more stable mRNA, and facilitating translation of target protein; (4) Changing the codon frequency of host preference, and increasing CAI index (codon adaptation index). The GC content in the nucleotide sequence is regulated by optimizing the nucleotide sequence of the RBD region of the S protein of the SARS-CoV-2Beta mutant strain and the nucleotide sequence of the RBD region of the S protein of the novel SARS-CoV-2Omicron mutant strain; simultaneously, the codon frequency of host preference is changed, and the CAI (codon adaptation index) index is improved; the free energy of the formed RNA secondary structure is reduced, the proportion of the Negative CIS element is reduced, the proportion of the repeated sequence in the sequence is reduced, in addition, the optimization of the signal peptide is carried out, and the specific algorithm of the company formed by the experiences of the inventor in the field for years is combined, so that the expression quantity of the RNA can be further improved, the optimized nucleotide sequence is obtained, and the RNA vaccine is prepared.
The optimization process comprises the following steps: the nucleotide sequence of the wild SARS-CoV-2Beta mutant strain S protein RBD region before optimization (EPI_ISL_860630, GISAID) is selected to be optimized according to the optimization strategy of the invention to obtain the nucleotide sequence shown as SEQ ID No.3, the nucleotide sequence of the wild novel SARS-CoV-2 Omicron mutant strain S protein RBD region before optimization (EPI_ISL_6640917, GISAID) is selected to be optimized according to the optimization strategy of the invention to obtain the nucleotide sequence shown as SEQ ID No.4, the nucleotide sequence of the coding signal peptide shown as SEQ ID No.7 is added to the front end N end of SEQ ID No.3, and the TGATAA stop codon is added to the end C end of SEQ ID No.4, so that the nucleotide sequence shown as SEQ ID No.5 is finally obtained, and the nucleotide sequence shown as SEQ ID No.9 is finally obtained by adopting the conventional commercial database (Integrated DNA Technologies, IDT) optimization strategy. Scoring the nucleotide sequence of SEQ ID No.5 before optimization and SEQ ID No.9 after optimization and obtained by conventional commercial optimization in the invention; in terms of optimizing and increasing expression of DNA sequences, key indexes of optimizing effect and DNA optimization are as follows: codon optimisation is indexed positively, GC content positively, negatively with the number of negative regulatory elements. As shown in fig. 1-3, the optimization strategy adopted by the invention is found to have a remarkable improvement on key indexes compared with the conventional commercial optimization strategy, and can be predicted to increase the expression efficiency of the optimized genes.
The wild sequence before the optimization, SEQ ID No.5 after the optimization of the invention and 3 nucleotide sequences of SEQ ID No.9 obtained from a conventional commercial database are respectively transformed and constructed into a pVAX1 vector (ThermoFisher, cat# V26020) to obtain 3 plasmid DNAs, namely pBeta-Omicron Dimer-wild, pBeta-Omicron Dimer and pBeta-Omicron Dimer-conventional optimization. 3 plasmids are respectively transfected into HEK293T cells for 48 hours, RNA is extracted, and the transcription level of plasmid DNA obtained in different optimization modes is identified by adopting a qPCR method. As shown in FIG. 4, the optimized DNA sequence of the invention can be increased by more than 150 times compared with the wild sequence before the optimization in RNA transcription level, and can be increased by more than 3 times compared with the conventional optimized molecules in the commercial database in RNA transcription level, so that the optimized nucleic acid molecules of the invention are better than the conventional commercial database. The improvement of the transcription level of the DNA vaccine can improve the protein expression quantity, so that the immune effect of the DNA vaccine is improved, the sequence obtained by the design of the invention is obviously improved in the transcription level, and the protein expression quantity is obviously improved, so that the obvious and better immune effect is obtained.
When designing and optimizing the nucleotide sequence, considering that the spatial structure folding of the antigen domain and the exposure degree of the antigen domain can be influenced by different protein domain tandem strategies, the more regular protein spatial structure can lead the antigen-antibody reaction to be better, and the more sufficient the exposure degree of the antigen domain is, the more complete the corresponding antibody reaction spectrum excited by the antigen domain is, and the better the immune effect can be. Therefore, the serial sequence of the SARS-CoV-2Beta mutant strain S protein RBD region (Beta RBD) and the novel SARS-CoV-2 Omicron mutant strain S protein RBD region (Omicron RBD) is analyzed by biological informatics means such as artificial intelligence binding protein structural analysis; as shown in FIG. 5, the analysis results show that the Beta-Omicron RBD cascade sequence (A diagram) and the Omicron-Beta RBD cascade sequence (B diagram) make the Beta RBD and the Omicron RBD two antigen domains exposed to similar degrees, but the Beta-Omicron RBD cascade sequence (A diagram) makes Beta-folding of the Beta RBD and the Omicron RBD two antigen domains on the space structure more regular, and the more regular protein space structure makes antigen-antibody reaction better, so that the Beta-Omicron RBD cascade strategy has better immune effect.
Example 2: construction process of DNA vaccine
1. Preparation method of coronavirus broad-spectrum candidate DNA vaccine
1.1. Construction of plasmids
The nucleotide sequence encoding the S protein RBD region of the novel SARS-CoV-2Beta mutant strain (derived from EPI_ISL_860630, GISAID) was designed as shown in SEQ ID No.3, and the nucleotide sequence encoding the S protein RBD region of the novel SARS-CoV-2 Omicron mutant strain (derived from EPI_ISL_6640917, GISAID) was designed as shown in SEQ ID No. 4. After SEQ ID No.3 and SEQ ID No.4 are directly connected in series, a nucleotide sequence of coding signal peptide shown as SEQ ID No.7 is added at the front end N end of SEQ ID No.3, and a TGATAA stop codon is added at the tail end C end of SEQ ID No.4, so that a nucleotide sequence shown as SEQ ID No.5 is finally obtained, and SEQ ID No.5 is inserted between BamHI and Xho I sites of the pVAX1 vector, so that a plasmid pBeta-Omicron Dimer is obtained.
The nucleotide sequence shown in SEQ ID No.8 was obtained by optimizing based on the wild-type sequence (MN 908947.3, NCBI) of the novel coronavirus SARS-CoV-2, and the nucleotide sequence shown in SEQ ID No.8 was inserted between BamHI and XhoI sites of the pVAX1 vector to obtain a novel coronavirus wild-type strain plasmid (pWT). The pWT wild strain vaccine is a product aiming at the wild strain in the early stage of the company, and currently enters phase III clinic, so that the vaccine has very excellent immune effect.
DNA vaccine sequence transformation
Mu.l of DH10B competent cell suspension was taken from a-80℃refrigerator and thawed on ice. Add plasmid DNA solution (volume no more than 10. Mu.l) and gently shake well and place on ice for 30min. The mixture was heat-shocked in a water bath at 42℃for 70 seconds and rapidly cooled on ice for 5min. 0.9ml of LB liquid medium (without antibiotics) is added into the tube, and after uniform mixing, the bacteria are cultured for 45min at 37 ℃ in a shaking way, so that the bacteria are recovered to a normal growth state. Shaking the bacterial liquid evenly, taking 100 mu L of the bacterial liquid, coating the bacterial liquid on a screening plate containing proper antibiotics, placing the bacterial liquid on the front surface upwards, inverting a culture dish after the bacterial liquid is completely absorbed by a culture medium, and culturing the bacterial liquid at 37 ℃ for 12-16h. The uniform shape of the monoclonal cells were selected, and the clones were pricked using a sterile pipette head and placed in 5mL of LB selection medium containing 50mg/mL kanamycin for overnight culture at 37 ℃.
DNA vaccine plasmid extraction
The bacterial liquid is added into 200-400 mL LB selection medium containing kanamycin (50 mg/mL mother liquor, 1:1000 is used) according to the ratio of 1:1000, and the bacterial liquid is cultured for 12-16h at 37 ℃ at 200 rpm. Plasmid extraction was performed with EndoFreen Plasmid Maxi kit (QIAGEN, germany): and (3) centrifuging the bacterial liquid cultured for 12-16h at the speed of 8000rpm at the temperature of 4 ℃ for 10min, collecting bacterial bodies by discarding the supernatant, adding 10ml of P1 Buffer to resuspension the bacterial liquid, adding 10ml of P2 Buffer, gently reversing for 4-6 times, uniformly mixing, and incubating at room temperature for 5min for full lysis. 10ml of P3 Buffer is added into the mixed solution, after 4-6 times of even mixing are gently reversed to terminate the cleavage, all the mixture is transferred into QIAfilter Cartridge, incubated for 10min at room temperature, and the embolic filtering supernatant is added. The filtrate was transferred to a clean endotoxin-free 50ml centrifuge tube, 2.5ml ER Buffer was added, gently inverted 10 times, mixed well and then placed on ice for incubation for 30min. QIAGEN-tip 500 was taken out, 10ml of QBT Buffer was added to the equilibrated column, the above liquid was transferred to the column, the plasmid was adsorbed by gravity flow, washed 2 times with 30ml of QC Buffer, and eluted with 15ml of QN Buffer. Each tube of sample was precipitated with 10.5ml of isopropanol and centrifuged at 4000g for 30min at 4 ℃. The supernatant was discarded, washed 1 time with 70% ethanol, and centrifuged at 4000g for 10min at 4 ℃. The supernatant was discarded, the pellet was dried, and 500. Mu.l of endotoxin-free water was added to each sample to resuspend the plasmid, thereby obtaining a recombinant expression plasmid for preparing DNA vaccine.
Example 3: mammalian cell transcriptional identification of coronavirus broad-spectrum candidate DNA vaccine
To verify whether the plasmid constructed in example 2 was able to be transcribed efficiently in mammalian cells, it was identified by methods of DNA transfection in vitro, RNA extraction, qPCR.
DNA vaccine in vitro transfection
Frozen HEK293T cell lines were removed from liquid nitrogen and centrifuged at 1000rpm for 5min after a 37 ℃ water bath to remove DMSO. Adding serum-free DMEM culture solution, washing once, adding into 5ml of DMEM culture solution containing 10% calf serum, 37deg.C and 5% CO 2 Culturing for 2-3 generations. After digestion of cells with pancreatin (0.25% EDTA) at 37℃for 1min and termination with complete medium, the cells were incubated at 2-4X 10 6 Cell/well density was plated on 60mm dishes, 5ml of growth medium (without 1% diabody) was added at 37℃with 5% CO 2 Culturing in an incubator for 24 hours.
Mu.g of pBeta-Omicron Dimer and 4. Mu.g of pWT were added to 500. Mu.l of reduced serum OPTI-MEM medium, respectively, and gently mixed, while 24. Mu.l of cationic liposome (Shanghai Saint, 40802ES 03) was gently mixed in 500. Mu.l of reduced serum OPTI-MEM medium, and left at room temperature for 5min, and the above two plasmids were mixed with liposome 1:1, respectively, and left at room temperature for 20min, to give a plasmid DNA/liposome complex.
The plasmid DNA/liposome complex was added to the 60mm dish for 24 hours at 37℃in 1 ml/dish, 5% CO 2 The incubators were incubated to 48 hours each for subsequent experiments.
2. Post-transfection RNA extraction
The cells transfected to 48 hours above were collected by digestion, resuspended in 1ml of complete medium, 100. Mu.l were aspirated for RNA extraction, and the remaining resuspension was used for subsequent WB sample preparation.
100 μl of the aspirated cell suspension was centrifuged at 4000rpm for 5min, the supernatant was discarded, and 350 μ l TRK Lysis Solution (containing 20% β -mercaptoethanol) was added to each for lysis. Each was then quenched with 350. Mu.l of 70% ethanol (prepared with DEPC water) and mixed by gun blowing.
Transferring the above mixture into HiBind RNA Column column, centrifuging 10000g for 1min, and discarding the filtrate. 500 μl Wash Buffer I was added to each column, and 10000g was centrifuged for 1min, and the filtrate was discarded. Each column was washed 2 times with 500. Mu.l Wash Buffer II, centrifuged at 10000g each for 1min, and the filtrate was discarded. The centrifuge speed was adjusted to the highest speed (17000 g) and centrifuged for 2min to volatilize the ethanol in the column. The column was transferred to a clean 1.5ml centrifuge tube Free of DNA and RNase, left at room temperature for 3-5min, after complete evaporation of ethanol, 50. Mu.l of RNase-Free Water was added to each, incubated for 5min at room temperature, and centrifuged at 17000g for 1min. The filtrate was aspirated again into the column, incubated for 5min at room temperature, and RNA was collected by centrifugation at 17000g for 1min and stored at-80 ℃.
RNA reverse transcription, qPCR reaction
RNA concentration was quantified by using an enzyme-labeled instrument (reading was performed using OD 260/280), and a solution was prepared according to the number of PCR required to be n (n=number of samples+1 tube negative control+1 tube positive control), and 10. Mu.l of a reaction system (2. Mu.l of 5X gDNA digester Buffer, 1. Mu.l of gDNA digestre, 100ng of RNA was prepared per sample, and RNase free ddH was used) 2 O was adjusted to 10. Mu.l in volume, gently mixed by blowing with a gun and incubated at 42℃for 2min. To each sample, 10. Mu.l of 2X Hifair II SuperMix plus was added, and after mixing with gentle blowing by a gun, incubation was performed at 25℃for 5min,42℃for 30min, and 85℃for 5 min. The collected cDNA was placed at-20℃for use.
The cDNA product obtained by reverse transcription is reacted according to qPCR kit. The reaction system is as follows:qPCR SYBR Green Master Mix (No Rox) 10. Mu.l each of the desired forward and reverse primers (forward primer: 5 'AAGCTGAACGACCTGGTTGCTTCA 3', SEQ ID No.10; reverse primer: 5'GGCAGCTTGTAGTTGTAG3', SEQ ID No. 11), 1. Mu.l each of the cDNA templates, and sterile ultra pure water were used to make up a total volume of 20. Mu.l. PCR reaction conditions: the total of 40 cycles of 95 ℃, 5min,95 ℃, 10s,56 ℃, 30s,72 ℃ and 30 s. The expression level of the target gene is 2 compared with that of the reference -△△C And (5) calculating a method.
Conclusion: as shown in FIG. 6, both the novel coronavirus broad-spectrum candidate DNA vaccine pWT and the coronavirus broad-spectrum candidate DNA vaccine pBeta-Omicron Dimer were able to promote transcription of antigen RNA at high levels in the empty vector (pVAX 1) after 48 hours of in vitro transfection, and the transcription level of coronavirus broad-spectrum candidate DNA vaccine pBeta-Omicron Dimer group RNA was significantly higher than that of pWT groups.
Example 4: identification of coronavirus broad-spectrum candidate DNA vaccine mammal cell antigen protein expression
To further verify whether the plasmid constructed in example 2 can be efficiently expressed in mammalian cells, it was identified by the Western Blot method by extracting the antigen protein.
1. Protein extraction
Plasmids pBeta-Omicron Dimer and pWT are respectively transfected into HEK293T cell lines, after 48 hours of transfection, the transfected culture solution is removed, the culture solution is washed once by precooled PBS, the PBS is removed, 150 μl of lysate (EDTA and protease inhibitor are added according to 1:100 before use) is added, and the mixture is blown for 10 times. Placed in a centrifuge at 12,000rpm at 4℃for 5 minutes. The supernatant was aspirated into 1.5mL centrifuge tubes, 50. Mu.L of supernatant was removed for each, 12.5. Mu.L of 5 Xprotein loading buffer was added, and the mixture was boiled in boiling water for 10min and then immediately removed for use.
2. Sample loading and SDS-PAGE electrophoresis
And adding 62.5 mu l of the supernatant sample after boiling and centrifugation into each hole of an SDS-PAGE gel, switching on a power supply, adjusting to constant voltage of 200V, and setting the time for 45min for electrophoresis. After the electrophoresis, SDS-PAGE was performed to prepare a transfer membrane. The PVDF membrane is taken and soaked in methanol for 30s for activation, and the PVDF membrane is placed in a 1X membrane transfer equilibrium solution for 1min.
3. Transfer film
Taking the positive electrode as the bottom surface according to the following steps: the eBlot L1 film-transferring gasket, the PVDF film, the gel and the eBlot L1 film-transferring gasket are sequentially overlapped, and the tube is used for removing interlayer bubbles once overlapped. Closing: PVDF membrane was removed from the flask and placed in a glass box containing 1 XTBST+5% skimmed milk powder and incubated in a shaker at 90rpm for 1h at room temperature. Washing: PVDF membrane was put into 1 XTBST and washed 3 times, 10 minutes each time, and the shaker was rotated at 90 rpm. Incubation resistance: PVDF membrane was placed in primary antibody solution (S-ECD/RBD monoclonal antibody (1), diluted 1:2000 with 1ug/ml XG014 anti) and incubated in shaker at 90rpm for 1h at room temperature. Washing: PVDF membrane was put into 1 XTBST and washed 5 times, 10 minutes each, and shaken at 90rpm in a shaker. Secondary antibody incubation: PVDF membrane was placed in secondary antibody solution (HRP conjugated Anti human IgG,1:5000 dilution and Goat Anti-Rabbit IgG Antibody (H & L) [ HRP ], pAb, 1:4000) and incubated for 1H at 90rpm at room temperature. Washing: PVDF membrane was put into 1 XTBST and washed 5 times, 10 minutes each time, and the shaker was rotated at 90 rpm. Color development: taking 3ml of chemiluminescent liquid A and 3ml of chemiluminescent liquid B according to the following ratio of 1: mixing at a ratio of 1, adding into PVDF membrane, incubating for 1-2min, and photographing.
Conclusion: as shown in FIG. 7, the novel coronal wild-type candidate DNA vaccine pWT and coronavirus broad-spectrum candidate DNA vaccine pBeta-Omacron Dimer were capable of expressing antigen proteins at high intracellular levels compared to empty vector (pVAX 1) after 48 hours of in vitro transfection.
Example 5: in vitro antigen protein expression verification of coronavirus broad-spectrum candidate DNA vaccine
To further verify that the expressed antigen was secreted extracellularly, plasmids pBeta-Omacron Dimer and pWT were transfected into HEK293T cell lines, respectively, in the same manner as the above experimental transfection method, and after 48 hours of transfection, supernatants were collected and assayed for secreted antigen proteins by ELISA.
Mono Rab TM SARS-CoV-2Neutralizing Antibody (BS-R2B 2), mAb, rabbit were diluted to 0.5ug/ml with coating buffer, added to ELISA plates at 100 ul/well, covered with sealing plate membrane and incubated overnight at 2-8deg.C. 1 XPBST was washed 3 times. 5% BSA (in PBST) was used as blocking solution, and after loading was completed at 100 ul/well, the wells were covered with a plate membrane and incubated at 37℃for 1h.1 XPBST was washed 3 times. The standard RBD (N501Y) protein was diluted to 100ng/ml with 3% BSA and subjected to 3-fold gradient dilution, 7 dilution gradients were set up, and 1 "0" well was additionally set up. Sample antigens (culture supernatant) were set up for a total of 4 gradients: stock solution, 10-fold dilution, 100-fold dilution and 1000-fold dilution. After all antigens were added to the plate at 100 ul/well, covered with a sealing plate membrane and incubated for 1h at 37 ℃.1 XPBST was washed 5 times. S-ECD/RBD monoclonal antibody (2) was diluted with 3% BSA at 1:80000, added to the plate at 100 ul/well, covered with a plate membrane, and incubated at 37℃for 1h. The wells were dried by washing 5 times with 1 XPBST at 250 ul/well. HRP Anti-Human IgG was diluted 1:5000 with 3% BSA, added to the plate at 100 ul/well, covered with plate membrane and incubated at 37℃for 1h.1 XPBST was washed 5 times. After mixing the TMB components at 1:1, the mixture was incubated at 100 ul/well for 10min at room temperature in the absence of light. 2M H2SO4 was taken and terminated at 50 ul/well. ELISA was placed in a microplate reader as prescribed to detect the absorbance at OD450 and OD620 at dual wavelengths.
Conclusion: as shown in FIG. 8, the coronavirus broad-spectrum candidate DNA vaccine pBeta-Omicron Dimer and the new coronavirus wild strain candidate DNA vaccine pWT can both express antigen proteins, and the pBeta-Omicron Dimer group is obviously higher than the pWT group, which indicates that the coronavirus broad-spectrum candidate DNA vaccine pBeta-Omicron Dimer has stronger antigen expression capability.
Example 6: broad-spectrum candidate DNA vaccine immunogenicity verification for coronavirus
To evaluate the immunogenicity of the vaccine prepared in example 2, as well as the effect of the immunization strategy on humoral and cellular immune responses, 6 week old C57BL/6 female mice without specific pathogens were purchased from Shanghai Laek and kept in Ai Diwei Xin Advaccine laboratories (Suzhou) animal facilities. Immunization against DNA vaccine: the DNA vaccine described in example 1 was injected into the anterior femur muscle sequentially according to different divided injections, followed by Electrical Pulsing (EP). An Electrical Pulse (EP) device consists of two sets of pulses with a constant current of 0.2 Amp. The second pulse set is delayed by 3 seconds. In each group there are two 52ms pulses with a delay between the pulses of 198ms. The first priming was counted as 0 day and the second immunization (booster immunization) was performed on day 14. Experimental grouping: (1) control vector plasmid pVAX 1. Mu.g; (2) experimental group wild-type strain DNA vaccine pWT μg; (3) The experimental group coronavirus broad-spectrum DNA vaccine pBeta-Omicron Dimer 2.5 mug; (4) The experimental group coronavirus broad-spectrum DNA vaccine pBeta-Omicron Dimer 10 mug; (5) The experimental group coronavirus broad-spectrum DNA vaccine pBeta-Omicron Dimer 25 mug; blood samples of mice were collected on days 14, 21, and 28, and specific antibodies in serum were measured. Day 14, 28, immunized mice were sacrificed to analyze cellular immune responses.
1. Assessment of antigen-specific humoral immune responses elicited by DNA vaccines
ELISA detection of antibody concentration
ELISA-based methods were used to evaluate RBD protein binding antibodies against SARS-CoV-2 wild-type strain (WT), SARS-CoV-2Delta mutant RBD protein binding antibodies, SARS-CoV-2 Omicron mutant RBD protein binding antibodies, 14 days after primary immunization and 14 days after booster immunization. Nunc 96-well ELISA plates were coated overnight at 4℃with 1. Mu.g/mL of SARS-CoV-2 wild-type strain RBD protein, 1. Mu.g/mL of SARS-CoV-2Delta mutant RBD protein, 1. Mu.g/mL of SARS-CoV-2 Omicron mutant RBD protein (Acro Biosystems, DE, USA), respectively. Plates were washed 3 times and then washed with PBS (0.0 containing 5% Bovine Serum Albumin (BSA)5% Tween 20, i.e., PBST buffer) was blocked for 1 hour at 37 ℃. Three times serial dilutions of mouse serum were added to each well and incubated for 1 hour at 37 ℃. The plates were washed five more times and then 1 was added at 37 ℃): after 8000-dilution goat anti-mouse IgG-HRP (GenScript, NJ, CN) incubation for 1 hour, the bound antibodies were subsequently detected. After the final wash, the plate was developed by using TMB substrate and washed with 50. Mu.l/well 2M H 2 SO 4 The reaction was terminated. The end point of the serum antibody titer was determined as the reciprocal of the highest dilution by reading at 450nm and 620nm, and the highest dilution of the sample was 2.1 times higher than the absorbance of the negative control (criterion: control (negative) OD450-620 value +.2.1, determination that the corresponding highest dilution at this OD value was serum antibody titer).
Conclusion: the results of the antibody detection are shown in fig. 9 and 10, and the coronavirus broad-spectrum candidate DNA vaccine pBeta-Omicron Dimer and the new coronavirus wild strain candidate DNA vaccine pWT can obviously excite the experimental animals to generate antigen-specific antibodies 14 days after the primary immunization and 14 days after the booster immunization. In the ELISA test, SARS-CoV-2 wild strain (WT) RBD protein, SARS-CoV-2Delta mutant strain RBD protein and SARS-CoV-2 Omicron mutant strain RBD protein are respectively adopted as in vitro coating antigens, and as shown in the result graph, the novel crown wild strain candidate DNA vaccine pWT can generate antibodies aiming at novel crown wild type antigens and antibodies aiming at Delta mutant strain antigens and Omicron mutant strain antigens, the pBeta-Omicron Dimer DNA vaccine provided by the invention also achieves quite remarkable technical effects, and the novel crown Omicron mutant strain antigens are better than the novel crown wild strain candidate DNA vaccine pWT in terms of equivalent dosages, and as mentioned above, pWT is a pre-product with excellent immune effect aiming at novel crown wild strain, so that the pBeta-Omicron Dimer DNA vaccine provided by the invention has better immunogenicity and broad spectrum.
1.2. Pseudo virus neutralizing antibody detection
Huh-7 cells were seeded in 96-well plates in DMEM containing 10% FBS for culture. To detect neutralizing antibody titers, mouse serum was serially diluted 1:2 in DMEM medium. Subsequently, the diluted serum samples were incubated with SARS-CoV-2 variant pseudoviruses at 37℃for 30 minutes and the mixture was added to Huh-7 cells for infection. After a further 4 hours incubation, the supernatant was replaced with fresh DMEM medium (containing 10% fbs). After 48 hours of further incubation, the cell supernatant was removed, and the absolute luciferin luminescence values in the lysed cells were detected using a firefly luciferase assay kit (Promega) and a microplate reader and relative values were calculated by normalizing to the virus control wells in the same plate. Neutralizing antibody titers were calculated using GraphPad Prism 9 and defined as the reciprocal of serum dilution (50% reduction in RLU compared to RLU in virus control wells after subtraction of background RLU in cell control wells).
Conclusion: as shown in FIG. 11, the coronavirus broad-spectrum candidate DNA vaccine pBeta-Omacron Dimer and the new coronavirus wild-strain candidate DNA vaccine pWT were able to neutralize the activity of viruses such as the new coronavirus wild strain (WT), beta mutant and Delta mutant on day 7 after booster immunization. As can be seen from the result graph, the pBeta-Omicron Dimer provided by the invention has more excellent effect than the novel crown wild strain candidate DNA vaccine pWT aiming at the novel crown Beta mutant strain virus, and pWT is a pre-product with excellent immune effect aiming at the novel crown wild strain, thus further more explaining that the pBeta-Omicron Dimer DNA vaccine provided by the invention has more excellent immunogenicity and broad spectrum.
1.3. Pseudo virus neutralizing antibody comparative experiments
Mice were immunized with 25. Mu.g of pBeta-Omicron Dimer DNA vaccine, 25. Mu.g of pWT DNA vaccine, 10. Mu.g of ZF2001 protein vaccine (recombinant protein vaccine from the market of ZF2001, from ZF 2001), and 25. Mu.g of ZF2001 DNA vaccine, and serum taken 14 days after booster immunization was used for detection of Omicron mutant pseudovirus neutralizing antibodies. The experimental method is consistent with the detection of the pseudo virus neutralizing antibody, and the data is counted in a geometric mean counting mode.
Conclusion: as shown in fig. 12, the coronavirus broad-spectrum candidate DNA vaccine pBeta-Omicron Dimer has better effect than the new coronavirus wild-strain candidate DNA vaccine ppt, ZF2001 protein vaccine, ZF2001 DNA vaccine against the new coronavirus Omicron mutant pseudovirus, as previously described, pWT is a pre-product with excellent immune effect against the new coronavirus wild strain, whereas ZF2001 is a recombinant protein vaccine which is currently marketed, ZF2001 DNA is a DNA vaccine corresponding to ZF2001 protein vaccine, thereby indicating that the pBeta-Omicron Dimer DNA vaccine of the present invention has significantly better immunogenicity against the new coronavirus Omicron mutant than ppt, ZF2001 DNA vaccine, etc., so that the vaccine of the present invention can be expected to provide excellent preventive protection against the new coronavirus Omicron mutant which is widely spread across multiple countries.
2. Further evaluation of DNA vaccine-elicited antigen-specific cellular responses
2.1 IFN-gamma ELISPot assay
Whether the DNA vaccine can promote cellular immunity was analyzed by ELISPot. Spleen cells were isolated 14 days after primary immunization and 14 days after booster immunization, and IFN-gamma positive cell ELISPot experiments were performed.
The mice were euthanized, spleened out and ground into single cell suspensions, 14 days after primary immunization and 14 days after booster immunization, respectively, in a sterile environment; cells were harvested by centrifugation, lysed after red blood cell lysate was resuspended, and PBS containing FBS stopped lysis; filtering, and counting the prepared single cell suspension; single cells were suspended in RPMI1640 medium supplemented with 10% fbs,1% penicillin/streptomycin. IFN-gamma assays were performed by using the mouse IFN-gamma ELISPot kit (Daidae, CN). Spleen cell suspensions of each mouse isolated by the above method were inoculated into each well coated with anti-IFN-gamma antibody at a density of 250,000 and CO at 37 ℃ 2 The culture chambers were stimulated with SARS-CoV-2 wild-type RBD peptide library, SARS-CoV-2Delta mutant RBD peptide library, and SARS-CoV-2 Omicron mutant RBD peptide library, respectively, for 20 hours. The operation is performed according to the product instruction. Culture medium and PMA/Iono served as negative and positive controls, respectively. Positive spots were quantitatively detected by iSpot Reader (AID, stra. Beta. Berg, germany). The Spot Formation Units (SFU) per million cells were calculated by subtracting the negative control wells.
Conclusion: IFN-. Gamma.ELISPot.As shown in FIG. 13, FIG. 14, the coronavirus broad-spectrum candidate DNA vaccine pBeta-Omacron Dimer and the novel coronavirus wild-strain candidate DNA vaccine pWT were effective in inducing high levels of antigen-specific IFN-. Gamma.responses both 14 days after primary immunization and 14 days after booster immunization. In the ELISPot test, the novel coronavirus wild-type RBD protein, the SARS-CoV-2Delta mutant RBD protein and the SARS-CoV-2 Omicron mutant RBD protein are respectively adopted as in vitro stimulating peptides, and when the novel coronavirus wild-type RBD protein is adopted as the stimulating peptide, the novel coronavirus wild-type nucleic acid vaccine pWT is beneficial, however, the coronavirus broad-spectrum candidate DNA vaccine pBeta-Omicron Dimer provided by the invention has a remarkably better effect than pWT in lower (10 mug) and same dose (25 mug) groups, and even under the dose which is obviously lower by 10 times (2.5 mug), the equivalent effect of pWT can be still achieved. Aiming at the novel crown wild type antigen, the novel crown Delta mutant strain antigen and the novel crown Omacron mutant strain antigen, the coronavirus broad-spectrum candidate DNA vaccine pBeta-Omacron Dimer provided by the invention has better effect than the novel crown wild type strain candidate DNA vaccine pWT. As described above, pWT is a pre-product with excellent immune effect against the novel crown wild strain, thereby further illustrating that the pBeta-Omicron Dimer DNA vaccine of the present invention has better immunogenicity and broad spectrum.
2.2 specific T cell immune response
The effect of vaccine-elicited antigen-specific cellular immune responses was further assessed, and spleen cells were isolated 14 days after primary immunization and 14 days after booster immunization, and flow cytometry detection experiments were performed.
Isolation of spleen cells: the mice were euthanized, spleened out and ground into single cell suspensions, 14 days after primary immunization and 14 days after booster immunization, respectively, in a sterile environment; cells were harvested by centrifugation, lysed after red blood cell lysate was resuspended, and PBS containing FBS stopped lysis; filtering, and counting the prepared single cell suspension; single cells were suspended in RPMI1640 medium supplemented with 10% fbs,1% penicillin/streptomycin.
Flow cytometry detection experiments: spleen cell suspension from each mouse obtained by the above method, 37 ℃,5% co 2 The following were stimulated with SARS-CoV-2 wild-type RBD peptide library (pool), SARS-CoV-2 Omicron mutant RBD peptide library or PMA/Iono, respectively, while using 1. Mu.gBrefeldin A blocking (BD, CA, USA) per ml for 6 hours. Spleen cells were stained for membrane surface and intracellular cytokines, stimulated spleen cells were stained with FVD-eFluor780, then washed, and stained with anti-mouse CD4 antibodies in the dark at room temperature for 30 minutes, respectively. Cells were permeabilized with a fixation/permeation buffer and stained for intracellular with anti-mouse IFN-gamma and TNF-alpha at 4℃for 45 min. Cells were washed twice and resuspended with 200 μl PBS, then collected using a flow cytometer (ThermoFisher, MA, usa), and then analyzed with FlowJo software (BD, CA, usa).
Conclusion: the results are shown in figures 15-18, and the generation of antigen specific CD4 ifnγ and CD4 TNF- α T cell subsets was significantly induced 14 days after primary immunization in the coronavirus broad-spectrum candidate DNA vaccine pBeta-Omicron Dimer alone different dose group compared to empty vector pVAX1 group, significantly better than pWT group, 14 days after primary immunization (figures 15 and 16). 14 days after boost (fig. 17 and 18), both the coronavirus broad-spectrum candidate DNA vaccine pBeta-Omicron Dimer and the new coronavirus wild-type candidate DNA vaccine pWT were able to significantly induce the production of antigen-specific CD4 ifnγ and CD4 TNF- α T cell subsets, and the coronavirus broad-spectrum candidate DNA vaccine pBeta-Omicron Dimer was elevated at lower doses (10 μg) and significantly better than pWT groups at the same dose (25 μg). Even at a significantly lower dose of 10 times (2.5 μg), a better effect than pWT can be achieved.
In the FACS test, the novel coronavirus wild-type RBD protein and the SARS-CoV-2 Omicron mutant RBD protein are respectively adopted as in vitro stimulating peptides, the novel coronavirus wild-type RBD protein and the SARS-CoV-2 Omicron mutant RBD protein are adopted as in vitro stimulating peptides, and the novel coronavirus wild-type RBD protein is obviously beneficial to the novel coronavirus wild-type nucleic acid vaccine pWT when being adopted as in vitro stimulating peptides, however, the coronavirus broad-spectrum candidate DNA vaccine pBeta-Omicron Dimer provided by the invention has obvious and better technical effects in 14 days after primary immunization and 14 days after booster immunization. Similarly, when SARS-CoV-2 Omicron mutant RBD protein was used as the in vitro stimulating peptide, the pBeta-Omicron Dimer of the present invention achieved significantly better technical results both 14 days after the initial immunization and 14 days after the booster immunization. Even at a significantly lower dose of 10 times (2.5 μg), a better effect than pWT can be achieved. As described above, pWT is a pre-product with excellent immune effect against the novel crown wild strain, thereby further illustrating that the pBeta-Omicron Dimer DNA vaccine of the present invention has better immunogenicity and broad spectrum.
From the results of examples 1 to 6, it is understood that the coronavirus broad-spectrum candidate DNA vaccine pBeta-Omicron Dimer of the present invention can not only be transcribed efficiently in mammalian cells, but also express and secrete the corresponding antigen proteins efficiently due to the efficient codon optimization system and reasonable sequence design; the pBeta-Omicron Dimer candidate DNA vaccine has immunogenicity and is expressed in humoral immunity and cellular immunity response, and the pBeta-Omicron Dimer candidate DNA vaccine can obviously excite experimental animals to generate antigen-specific antibodies on the 14 th day after primary immunity and the 7 th day after booster immunity and the 14 th day, so that antibodies aiming at new crown wild type antigens can be generated, antibodies aiming at new crown Delta mutant antigens, beta mutant antigens and Omicron mutant antigens can be generated, and the vaccine has neutralization activity; for cellular immune responses, the pBeta-Omicron Dimer DNA vaccine is capable of inducing not only high levels of antigen-specific IFN-gamma responses, but also the production of antigen-specific CD4 ifnγ and CD4 TNF-alpha T cell subsets against the novel crown wild-type antigen, novel crown Delta mutant antigen, and omacron mutant antigen; both humoral immunity and cellular immunity experiments show that the pBeta-Omicron Dimer DNA vaccine has good immunogenicity and broad spectrum.
Notably, the pWT wild strain vaccine is a product of the company aiming at SARS-CoV-2 wild strain in the early stage, and has very excellent immune effect in the phase III clinic. In the above experiments, for example ELISA, pseudovirus neutralization, ELISPOT and FACS detection, not only the novel coronaSARS-CoV-2 wild-type RBD protein or virus was used as an in vitro coating antigen or stimulating peptide, but also the SARS-CoV-2Delta mutant strain RBD protein or virus and the SARS-CoV-2 Omicron mutant strain RBD protein or virus were used as in vitro coating antigens or stimulating peptides for detection of antigen-specific immune responses, and in the neutralization experiments, in addition to the above three viruses, the novel coronabeta mutant strain virus was used for experimental detection. Even when the conditions are obviously more favorable for the novel crown wild type nucleic acid vaccine pWT (the novel crown SARS-CoV-2 wild type RBD protein is adopted), the pBeta-Omicron Dimer DNA vaccine provided by the invention achieves obvious technical effects and even has obviously better effects than the novel crown wild type nucleic acid vaccine pWT, in particular to the cellular immunity level (fig. 13-18), and further illustrates that the pBeta-Omicron Dimer DNA vaccine provided by the invention has better immunogenicity and broad spectrum. When the RBD protein or virus of SARS-CoV-2Delta mutant strain and the RBD protein or virus of SARS-CoV-2 Omicron mutant strain are used as the in vitro coating antigen or stimulating peptide, the pSARS-Omicron Dimer DNA vaccine of the invention obviously induces higher humoral immunity and cellular immunity level than pWT.
Meanwhile, ZF2001 is a recombinant protein vaccine which is marketed at present, ZF2001 DNA is a DNA vaccine aiming at the ZF2001 protein vaccine, and in a pseudo-virus neutralization experiment aiming at an Omicron mutant strain, the pBeta-Omicron Dimer DNA vaccine provided by the invention is obviously superior to pWT, the ZF2001 protein vaccine and the ZF2001 DNA vaccine, so that the pBeta-Omicron Dimer DNA vaccine provided by the invention has better immunogenicity aiming at a new crown Omicron mutant strain virus. Thus, it is expected that the vaccine of the present invention can provide excellent preventive protection against a novel crown Omicron mutant which is widely spread in many countries and regions around the world.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. A recombinant nucleic acid molecule comprising any one of (a) to (c) as follows:
(a) The recombinant DNA molecules comprise a first DNA molecule encoding the S protein RBD region of the novel SARS-CoV-2Beta mutant strain and a second DNA molecule encoding the S protein RBD region of the novel SARS-CoV-2Omicron mutant strain;
(b) A DNA molecule derived from (a) by substitution, deletion or addition of one or several nucleotides in the nucleotide sequence of the recombinant DNA molecule defined in (a) and having the function of encoding the S protein RBD region of the novel SARS-CoV-2Beta mutant strain and encoding the S protein RBD region of the novel SARS-CoV-2Omicron mutant strain;
(c) Nucleic acid molecules which hybridize under stringent conditions with the nucleotide sequence of the recombinant DNA molecule defined in (a) or the DNA molecule defined in (b) and which encode the S protein RBD region of the novel SARS-CoV-2Beta mutant strain and the S protein RBD region of the novel SARS-CoV-2Omicron mutant strain.
2. The recombinant nucleic acid molecule of claim 1, wherein the first DNA molecule encodes an amino acid sequence as set forth in SEQ ID No. 1; the amino acid sequence of the second DNA molecule is shown as SEQ ID No. 2.
3. The recombinant nucleic acid molecule of claim 2, wherein the nucleotide sequence of the first nucleic acid molecule is set forth in SEQ ID No. 3; the nucleotide sequence of the second nucleic acid molecule is shown as SEQ ID No. 4;
Preferably, the nucleotide sequence of the recombinant nucleic acid molecule is the nucleotide sequence shown as SEQ ID No.5 or has at least 90% identity with the nucleotide sequence shown as SEQ ID No.5 and encodes the RBD region of the S protein of the novel SARS-CoV-2Beta mutant strain and the RBD region of the S protein of the novel SARS-CoV-2Omicron mutant strain.
4. A biomaterial, comprising:
a construct comprising the recombinant nucleic acid molecule of any one of claims 1 to 3; and, a third nucleic acid molecule encoding a signal peptide linked to the 5' end of the recombinant nucleic acid molecule;
(ii) recombinant expression vectors, including original expression vectors; and, inserting into the original expression vector a coding nucleic acid fragment selected from the group consisting of the recombinant nucleic acid molecule of any one of claims 1 to 3 or the (i) construct; preferably, the original expression vector is a pVAX1 plasmid;
(iii) a transformant obtained by introducing the recombinant expression vector of (ii) into a host cell selected from the group consisting of insect cells, yeast, avian cells, and mammalian cells; preferably, the host cell is HEK293, CHO or COS-7;
(iv) a polypeptide comprising a polypeptide encoded by the recombinant nucleic acid molecule of any one of claims 1 to 3, a polypeptide encoded by the construct of (i), or a polypeptide obtained by expression of a transformant of (iii);
(V) an antibody that specifically binds to the (IV) polypeptide.
5. The biological material of claim 4, wherein the signal peptide encoded by the third nucleic acid molecule comprises the following (c) or (d):
(c) A signal peptide with an amino acid sequence shown as SEQ ID No. 6;
(d) A signal peptide derived from (c) having a signal peptide function by substituting, deleting or adding one or more amino acids in the amino acid sequence of the signal peptide defined in (c).
6. Use of a recombinant nucleic acid molecule according to any one of claims 1 to 3 or a biological material according to any one of claims 4 to 5 in (a) or (B) as follows:
(A) Preparing vaccine for preventing and/or treating SARS-CoV-2 virus infection;
(B) Preparing medicine for preventing and/or treating SARS-CoV-2 virus related diseases.
7. The use according to claim 6, wherein the SARS-CoV-2 virus comprises a wild-type strain, a B.1.617.2 mutant, a B.1.1.7 mutant, a B.1.351 mutant, a P.1 mutant, a B.1.2 mutant, a B.1 mutant, a B.1.621 mutant, a B.1.525 mutant, a B.1.526 mutant, a C.37 mutant, a B.1.617.1 mutant or a B.1.1.529 mutant.
A DNA vaccine comprising the recombinant nucleic acid molecule of any one of claims 1 to 3 or the recombinant expression vector of any one of claims 4 to 5.
9. The DNA vaccine of claim 8, further comprising at least one of a pharmaceutically acceptable adjuvant, carrier, diluent, or excipient;
and/or at least one drug having a therapeutic effect on SARS-CoV-2 virus;
preferably, the adjuvant comprises at least one of an aluminium adjuvant, TLRs ligand, metal ion, cytokine or chemokine adjuvant;
further preferably, the metal ion comprises Mn 2+ And/or Zn 2+ 。
10. The method for producing a DNA vaccine according to claim 8 or 9, comprising introducing the recombinant nucleic acid molecule according to any one of claims 1 to 3 or the recombinant expression vector according to any one of claims 4 to 5 into a host cell, culturing the host cell, and extracting the recombinant nucleic acid molecule or the recombinant expression vector from the host cell.
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