CN114573667B

CN114573667B - Mutant strain DNA vaccine of SARS-CoV-2 virus B.1.1.529 and application thereof

Info

Publication number: CN114573667B
Application number: CN202210485326.2A
Authority: CN
Inventors: 赵干; 张志宇; 侯佳望; 丁媛; 俞庆龄
Original assignee: Advaccine Suzhou Biopharmaceutical Co ltd
Current assignee: Advaccine Suzhou Biopharmaceutical Co ltd
Priority date: 2022-05-06
Filing date: 2022-05-06
Publication date: 2022-08-02
Anticipated expiration: 2042-05-06
Also published as: CN114573667A

Abstract

The invention relates to the field of biotechnology, and particularly provides a SARS-CoV-2 virus B.1.1.529 mutant strain DNA vaccine and application thereof. The invention provides SEQ ID NO: the 1 sequence can be efficiently transcribed and expressed in a eukaryotic expression system, and has immunogenicity which is shown in humoral immunity and cellular immunity response, and the nucleic acid vaccine taking the sequence as an active component also has good immunogenicity.

Description

Mutant strain DNA vaccine of SARS-CoV-2 virus B.1.1.529 and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to a DNA molecule for coding SARS-CoV-2 virus B.1.1.529 mutant antigen, a DNA vaccine and application thereof.

Background

SARS-CoV-2 is a single-stranded positive-strand RNA virus with an envelope structure, which is highly susceptible to mutation. Among these, the presently emerging mutant Ormck Ron (Invitron: Omicron, accession number: B.1.1.529) has 32 mutations in the spike protein S protein, 15 mutations to the Receptor Binding Domain (RBD) region, including K417N, S477N, T478K, E484K, N501Y, G339D, S371L, S373P, S375F, N440K, G446S, Q493R, G496S, Q498R, and Y505H (Wu Jie, Liu Tu, Liumin, etc. Chinese general medicine, 2022) and has important amino acid mutation sites for Alpha (Alpha), Beta (Beta), Gamma (Gamma), and Delta (Delta) spike proteins, e.g., N501Y mutations in Alpha and Beta mutant strains, which increase the ability of the Alpha and Beta (ACE) spike protein to bind to vasopressin 2 and thereby increase the ability of the vasopressin 36501 protein to convert the vasopressin (ACE) enzyme proteins, 2; the E484K mutation, which occurs in beta and gamma mutants, can help variant strains to escape from the key sites of current vaccine immunity; in addition, the B.1.1.529 mutant rarely has two mutations near the furin cleavage site, namely P681H and N679K, which may enhance the immune escape capacity of the mutant, so that the spike protein is easier to be cleaved, thereby enhancing the virus infectivity. Thus, there is a need to develop more effective vaccines against mutant strains.

Disclosure of Invention

The Ormckhun mutant strain of the present invention is SARS-CoV-2 virus mutant strain with the number B.1.1.529.

It is an object of the present invention to provide a DNA molecule encoding the mutant antigen of SARS-CoV-2 virus B.1.1.529, which alleviates at least one of the technical problems of the prior art.

Another object of the present invention is to provide a biomaterial comprising the DNA molecule.

The third purpose of the invention is to provide the application of the biological material.

The fourth purpose of the invention is to provide a SARS-CoV-2 virus B.1.1.529 mutant DNA vaccine containing the DNA molecule.

The fifth object of the present invention is to provide a method for producing the DNA vaccine.

The sixth object of the present invention is to provide the use of the above DNA vaccine.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the invention provides a DNA molecule for coding SARS-CoV-2 virus B.1.1.529 mutant strain antigen, the DNA molecule has the nucleotide sequence shown in SEQ ID NO: 1, or a nucleotide sequence corresponding to SEQ ID NO: 1 has a nucleotide sequence of at least 90% identity.

The present invention also provides a biomaterial comprising:

(a) recombinant expression vectors comprising the DNA molecules described above;

(b) a cell comprising the DNA molecule of (a) or the recombinant expression vector of (a);

(c) a polypeptide encoded by the DNA molecule described above.

Further, the recombinant expression vector comprises a eukaryotic expression vector, and the eukaryotic expression vector comprises pVAX 1.

Preferably, the cells include HEK293, CHO, COS-7, and the like.

The invention also provides the application of the DNA molecule or the biological material in the following (A) or (B):

(A) preparing a vaccine for preventing and/or treating SARS-CoV-2 virus infection;

(B) preparing the medicine for preventing and/or treating the related diseases caused by SARS-CoV-2 virus.

Furthermore, the SARS-CoV-2 virus comprises a mutant B.1.1.529, a wild strain, a mutant B.1.1.7, a mutant B.1.351, a mutant P.1, a mutant B.1.2, a mutant B.1, a mutant B.1.525, a mutant B.1.526, a mutant C.37, a mutant B.1.617.1 or a mutant B.1.617.2.

The invention also provides a SARS-CoV-2 virus B.1.1.529 mutant strain DNA vaccine, which comprises the DNA molecule.

Further, the DNA molecule is present in a recombinant expression vector comprising pVAX 1.

Further, the DNA vaccine also comprises 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 an aluminium adjuvant and/or a TLRs ligand and/or a metal ion such as Mn ²⁺ 、Zn ²⁺ And/or cytokine and/or chemokine adjuvants, and the like.

The invention also provides a preparation method of the DNA vaccine, which comprises the steps of introducing the recombinant vector containing the DNA molecule into host cells, culturing, and extracting the recombinant vector in the host cells to obtain the DNA vaccine.

In addition, the invention also provides the application of the DNA vaccine, which comprises the following (i) to (iii):

(i) regulating the immune function of the organism;

(ii) anti-SARS-CoV-2 virus infection;

(iii) prevention of immunopathological damage;

the SARS-CoV-2 virus includes B.1.1.529 mutant, wild strain, B.1.1.7 mutant, B.1.351 mutant, P.1 mutant, B.1.2 mutant, B.1 mutant, B.1.525 mutant, B.1.526 mutant, C.37 mutant, B.1.617.1 mutant or B.1.617.2 mutant.

Compared with the prior art, the invention has the following beneficial effects:

the invention optimizes the coding DNA sequence of the mutant Spike protein of SARS-CoV-2 virus B.1.1.529 by using different optimization principles to obtain the protein with the sequence shown in SEQ ID NO: 1 or a nucleotide sequence corresponding to SEQ ID NO: 1 has a nucleotide sequence of at least 90% identity. The DNA molecule can efficiently transcribe and express the mutant Spike antigen of SARS-CoV-2 virus B.1.1.529, has immunogenicity, and can induce specific humoral immunity and cell immunity response.

Based on the beneficial effects of the DNA molecule coding the mutant strain antigen of SARS-CoV-2 virus B.1.1.529, the invention also provides a DNA vaccine comprising the above DNA molecule. The DNA vaccine can be effectively transcribed and expressed in mammalian cells, has good immunogenicity, and can remarkably stimulate experimental animals to generate antigen specific antibodies 7 days after the enhanced immunity for humoral immune response, so that the DNA vaccine not only can generate antibodies aiming at the wild antigen of the new crown, but also can generate antibodies aiming at the mutant antigen of the new crown B.1.1.529; for cellular immune response, the DNA vaccine can induce high-level new crown wild type antigen specific IFN-gamma reaction, can also induce high-level new crown B.1.1.529 mutant strain antigen and new crown Delta (Delta) mutant strain antigen specific IFN-gamma reaction, and reflects the good immunogenicity and broad spectrum of the DNA vaccine.

Based on the DNA vaccine, the DNA vaccine provided by the invention can adjust the immune function of organisms and effectively prevent

Infection of SARS-CoV-2 virus and its mutant strain, especially B.1.1.529 mutant strain, and also can be used for preventing and treating diseases caused by SARS-CoV-2 virus and its mutant strain, especially B.1.1.529 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 used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph showing the result of codon optimization index scoring of a DNA sequence encoding a Spike protein of the B.1.1.529 mutant strain;

FIG. 2 is a graph showing the results of GC content scoring after optimization of the DNA sequence encoding the Spike protein of the B.1.1.529 mutant strain;

FIG. 3 is a graph showing the result of scoring the number of negative regulatory elements after optimizing the DNA sequence encoding the Spike protein of the B.1.1.529 mutant strain;

FIG. 4 is a graph of the results of qPCR fold expression after optimization of the DNA sequence encoding the Spike protein of the B.1.1.529 mutant strain;

FIG. 5 shows the qPCR expression result of the new crown B.1.1.529 mutant strain candidate DNA vaccine provided by the embodiment of the invention;

FIG. 6 shows the Western Blot detection result of antigen protein of the new crown B.1.1.529 mutant strain candidate DNA vaccine provided by the embodiment of the invention;

FIG. 7 shows the results of antigen-specific antibodies at day 7 after boosting immunization of the candidate DNA vaccines of the new crown wild strain and the mutant B.1.1.529 provided by the embodiment of the invention;

FIG. 8 shows the neutralizing antibody results of the candidate DNA vaccine of the new crown wild strain, B.1.1.529 mutant strain provided by the embodiment of the invention on the 7 th day after the boosting immunization;

FIG. 9 shows the results of antigen-specific IFN-. gamma.ELISOPT at day 14 after boosting immunization with the candidate DNA vaccines of the new crown wild strain and the B.1.1.529 mutant strain provided in the embodiment of the present invention;

FIG. 10 shows the results of IFN-. gamma.ELISOPT specific to Delta mutant antigen at 14 days after B.1.1.529 mutant strain candidate DNA vaccine provided in the examples of the present invention was boosted.

Detailed Description

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" and other forms is not limiting.

Generally, the nomenclature used, and the techniques thereof, in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly employed in the art.

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

According to one aspect of the invention, the invention optimizes the coding DNA sequence of the mutant Spike protein of SARS-CoV-2 virus B.1.1.529 by using different optimization principles to obtain the protein with the sequence shown in SEQ ID NO: 1 or a nucleotide sequence corresponding to SEQ ID NO: 1 has a nucleotide sequence of at least 90% identity. The DNA molecule can be transcribed efficiently, is more beneficial to efficiently expressing the SARS-CoV-2 virus B.1.1.529 mutant Spike antigen in a eukaryotic expression system, has good immunogenicity, and can induce specific humoral immunity and cellular immune response.

It is understood that, in the present invention, "identity" refers to similarity between nucleotide sequences, including nucleotide sequences that are identical to SEQ ID NO: 1 (e.g., can be, but is not limited to, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identical.

Optionally, the optimization also comprises replacing the SARS-CoV-2 virus B.1.1.529 mutant gene signal peptide with a high-efficiency expression signal peptide, thereby improving the expression efficiency of the DNA sequence in the host.

The invention also provides biological materials related to the DNA molecules:

(a) recombinant expression vectors comprising the DNA molecules provided by the invention. Wherein the vector can be a eukaryotic expression vector, and the protein encoded by the DNA molecule is generated through a cell transcription and translation mechanism. Alternatively, the 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 a transcription termination sequence. Preferably, the eukaryotic expression vector includes pVAX1, but is not limited to any other expression vector capable of expressing DNA and enabling a cell to translate sequences into antigens recognized by the immune system.

(b) The cell is obtained by introducing the DNA molecule provided by the invention or the recombinant expression vector of (a) into a host cell. The host cell may be a eukaryotic cell, typically an insect cell, a yeast cell, an avian cell, or a mammalian cell, among others as appropriate. The cells include HEK293, CHO, COS-7 and the like.

(c) Polypeptides encoded by the DNA molecules provided by the invention. Based on the polypeptide, an antibody, such as a monoclonal antibody or a polyclonal antibody, capable of specifically binding thereto may also be provided.

It can be understood that the biological material provided by the invention can be directly applied to the production of different requirements and scenes as a biological module.

According to another aspect of the present invention, the present invention also provides the use of the above DNA molecule or biomaterial in the preparation of a vaccine for preventing and/or treating SARS-CoV-2 virus infection, and/or in the preparation of a medicament for preventing and/or treating related diseases caused by SARS-CoV-2 virus, such as lung injury, brain injury, liver and kidney injury, and heart injury.

Preferably, the SARS-CoV-2 virus comprises a mutant B.1.1.529 strain, a wild strain, a mutant B.1.1.7 strain, a mutant B.1.351 strain, a mutant P.1 strain, a mutant B.1.2 strain, a mutant B.1 strain, a mutant B.1.525 strain, a mutant B.1.526 strain, a mutant C.37 strain, a mutant B.1.617.1 strain or a mutant B.1.617.2 strain.

Based on the beneficial effects of the DNA molecule coding the mutant strain antigen of SARS-CoV-2 virus B.1.1.529, the invention also provides a DNA vaccine comprising the above DNA molecule.

The DNA vaccine can effectively transcribe and express the mutant Spike antigen of the SARS-CoV-2 virus B.1.1.529 in mammalian cells, more efficient immune reaction is excited, and for humoral immune response, the DNA vaccine can obviously excite an experimental animal to generate antigen specific antibody 7 days after strengthening immunity, not only can generate antibody aiming at the wild antigen of the new crown, but also can generate antibody aiming at the mutant antigen of the new crown B.1.1.529; for cellular immune response, the DNA vaccine can induce high-level new crown wild type antigen specific IFN-gamma reaction, can also induce high-level new crown B.1.1.529 mutant strain antigen and new crown Delta (Delta) mutant strain antigen specific IFN-gamma reaction, and reflects the good immunogenicity and broad spectrum of the DNA vaccine.

In some embodiments, the DNA vaccine further comprises a pharmaceutically acceptable adjuvant, carrier, diluent or excipient to increase the acceptance of its active ingredient DNA moleculeThe ability of the subject to mount an immune response. Wherein the pharmaceutically acceptable adjuvant may be selected from aluminium adjuvants and/or TLRs ligands and/or metal ions such as Mn ²⁺ 、Zn ²⁺ And/or cytokine and/or chemokine adjuvants, and the like.

In other embodiments, the DNA vaccine further comprises at least one drug that has a therapeutic effect on SARS-CoV-2 virus to enhance the therapeutic effect of the vaccine on the associated disease caused by SARS-CoV-2 virus.

The action mechanism of the DNA vaccine provided by the invention is as follows: the SARS-CoV-2 virus B.1.1.529 mutant surface antigen Spike antigen coding DNA is optimized by different optimization algorithms, and its wild type gene signal peptide is replaced by high-efficiency expression signal peptide, and then inserted into eukaryotic expression vector, and introduced into host cell to make it high-efficiency express virus Spike antigen in host cell, and through antigen presentation process systematically activate antiviral humoral immune response and cellular immune response. Antibodies generated by the activated humoral immune response can prevent viral entry, and the activated cellular immune response can 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 the application of the DNA vaccine, which comprises the following steps:

(i) regulating the immune function of the organism;

(ii) anti-SARS-CoV-2 virus infection;

(iii) preventing immunopathological damage.

The invention is further illustrated by the following examples. The materials in the examples are prepared according to known methods or are directly commercially available, unless otherwise specified.

Example 1: optimized screening of nucleic acids encoding S proteins

In order to increase the protein expression of the target protein in the 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 the degenerate codon according to the preference of the host cell to the nucleic acid codon, so that the optimized sequence contains more nucleic acid codons which are beneficial to the recognition of the host cell; (2) further optimizing the GC content in the nucleic acid sequence on the basis of codon preference optimization, so that the sequence with the optimized GC content can express more target proteins; (3) optimizing the nucleic acid sequence to make it able to transcribe more stable mRNA, facilitating translation of the target protein; (4) the host-biased codon frequency was changed to increase the CAI index (codon adaptation index). The application optimizes the coding nucleotide sequence of the surface protein Spike of the SARS-CoV-2 virus B.1.1.529 mutant strain, and adjusts the GC content in the nucleotide sequence; meanwhile, the codon frequency of host preference is changed, and the CAI (codon adaptation index) index is improved; the method has the advantages of reducing the free energy for forming an RNA secondary structure, reducing the proportion of Negative CIS elements, reducing the proportion of repeated sequences in the sequence, optimizing signal peptides, and combining with a company-specific algorithm formed by the inventor through years of experience in the field, so that the expression quantity of the nucleic acid vaccine can be further improved, an optimized nucleotide sequence is obtained, and the nucleic acid vaccine is prepared.

The optimization process comprises the following steps: selecting a wild B.1.1.529 mutant strain Spike (S protein) sequence (EPI _ ISL _6640917, GISAID) before optimization as an antigen sequence, and obtaining the sequence shown in SEQ ID NO: 1, and obtaining the nucleotide sequence shown in SEQ ID NO: 3. For the optimized wild B.1.1.529 sequence and the optimized SEQ ID NO: 1 and the conventional commercial optimization of SEQ ID NO: 3 is scored; in the aspect of optimizing and increasing expression of DNA sequences, key indexes of optimizing effect and DNA optimization are as follows: codon optimization indices are positively correlated, GC content is positively correlated, and the number of negative regulatory elements is negatively correlated. As shown in FIGS. 1-3, the results show that after the B.1.1.529 mutant strain Spike sequence is optimized, the optimization strategy adopted by the invention is found to be obviously improved in key indexes compared with the conventional commercial optimization strategy, and the optimization strategy adopted by the invention can be predicted to increase the expression efficiency of the optimized gene.

The mutant strain B.1.1.529 is optimized into a wild sequence and an optimized sequence shown in SEQ ID NO: 1 and SEQ ID NO: 3 into pVAX1 vector (ThermoFisher, cat # V26020) to obtain 3 kinds of plasmid DNA, pB.1.1.529-wild, pB.1.1.529 and pB.1.1.529-optimized conventionally. 3 plasmids are respectively transfected into a HEK293T cell strain for 48h, RNA is extracted, and the transcription level of plasmid DNA obtained by different optimization modes is identified by adopting a qPCR method. The results are shown in FIG. 4, the optimized B.1.1.529 mutant DNA sequence of the invention can be increased by more than 300 times at the RNA transcription level compared with the wild sequence before optimization, and can be increased by more than 3 times at the RNA transcription level compared with the molecule conventionally optimized in the commercial database, further demonstrating that the optimized nucleic acid molecule of the invention is superior to the molecule conventionally obtained in the commercial database. The improvement of the transcription level of the DNA vaccine can improve the protein expression quantity, thereby improving the immune effect of the DNA vaccine, the sequence designed and obtained by the invention is obviously improved in the transcription level, the protein expression quantity is also obviously improved, and the obvious better immune effect is obtained.

Example 2: construction process of DNA vaccine

1. Preparation method of new coronavirus candidate DNA vaccine

1.1. Construction of plasmids

As described in example 1, the mutant b.1.1.529 sequence (EPI _ ISL _6640917, GISAID) was optimized to obtain the sequence shown in SEQ ID NO: 1, the nucleotide sequence shown as SEQ ID NO: 1 is inserted between BamH I and Xho I sites of pVAX1 vector to obtain new mutant strain plasmid pB.1.1.529 of coronavirus B.1.1.529.

Optimization based on the new crown wild type sequence (MN 908947.3, NCBI) gave SEQ ID NO: 2, the nucleotide sequence shown as SEQ ID NO: 2 is inserted between BamH I and Xho I sites of pVAX1 vector to obtain new coronavirus wild strain plasmid (pWT). pWT the wild strain vaccine is a product aiming at the wild strain at the early stage of the company, is about to enter the phase III clinic at present, and has very excellent immune effect.

DNA vaccine sequence transformation

From a-80 ℃ freezer, 100. mu.L of DH10B competent cell suspension was removed and thawed on ice. Add plasmid DNA solution (volume not more than 10 u L) gently shake, ice placed for 30 min. The mixture was heated in a water bath at 42 ℃ for 70 seconds and rapidly cooled on ice for 5 min. 0.9mL of LB liquid medium (containing no antibiotics) was added to the tube, mixed well and cultured with shaking at 37 ℃ for 45min to restore the bacteria to normal growth state. Shaking the bacterial liquid uniformly, coating 100 μ L of the bacterial liquid on a screening plate containing appropriate antibiotics, placing the bacterial liquid with the front side upward, inverting the culture dish after the bacterial liquid is completely absorbed by the culture medium, and culturing for 12-16h at 37 ℃. The single-clone cells with uniform shapes were selected, and the colonies were picked up by using a sterile pipette tip and then cultured overnight at 37 ℃ in 5mL of LB selection medium containing 50mg/mL of kanamycin.

DNA vaccine plasmid extraction

The above-mentioned bacterial suspension was added to 200-400mL LB selection medium containing kanamycin (50 mg/mL of mother liquor, 1:1000 used) at 1:1000 and cultured at 37 ℃ at 200rpm for 12-16 hours. Plasmid extraction was performed with an EndoFreen Plasmid Maxi kit (QIAGEN, Germany): centrifuging the bacterial liquid cultured for 12-16h at 8000rpm for 10min at 4 ℃, removing supernatant, collecting bacterial bodies, adding 10mL of Buffer P1 heavy-suspension bacterial liquid, adding 10mL of Buffer P2, slightly reversing for 4-6 times, mixing uniformly, and incubating at room temperature for 5min for sufficient lysis. 10mL Buffer P3 was added to the mix, after terminating lysis by gently inverting 4-6 times of the mix, all were transferred to a QIAfilter Cartridge, incubated at room temperature for 10min, and the supernatant was filtered by adding plug. The filtrate was transferred to a clean endotoxin-free 50mL centrifuge tube, 2.5mL Buffer ER was added, the mixture was mixed by gentle inversion 10 times and incubated on ice for 30 min. The QIAGEN-tip 500 was removed and added to a 10mL Buffer QBT equilibrated column, and the above liquid was transferred to the column, and the plasmid was adsorbed by gravity flow, washed 2 times with 30mL Buffer QC, and eluted with 15mL Buffer QN. Samples from each tube were precipitated with 10.5mL of isopropanol and centrifuged at 4000g for 30min at 4 ℃. The supernatant was discarded, washed with 70% ethanol 1 time, centrifuged at 4000g for 10min at 4 ℃. Abandoning the supernatant, air-drying the precipitate, and adding 500 mu L of endotoxin-free water into each sample to carry out resuspension on the plasmid so as to obtain the DNA vaccine plasmid.

Example 3: mammalian cell transcriptional identification of new coronavirus candidate DNA vaccines

To verify whether the plasmid constructed in example 2 can be transcribed efficiently in mammalian cells, it was identified by methods of in vitro transfection of DNA, extraction of RNA, and qPCR.

1. DNA vaccine in vitro transfection

The frozen HEK293T cell line was removed from the liquid nitrogen and DMSO was removed by centrifugation at 1000rpm for 5 minutes after a 37 ℃ water bath. Washing with serum-free DMEM medium at 37 deg.C and 5% CO in 5mL of 10% calf serum-containing DMEM medium ₂ Culturing for 2-3 generations. The cells were digested with pancreatin (containing 0.25% EDTA) at 37 ℃ for 1min and stopped with complete medium, and then incubated at 2-4X 10 ⁶ The density of cells/well was plated on a 60mm dish and 5mL growth medium (without 1% double antibody) was added at 37 ℃ with 5% CO ₂ Culturing in an incubator for 24 h.

Mu.g of sterile plasmid pB.1.1.529 was added to 500. mu.L of serum-free OPTI-MEM medium, and gently mixed, while 24. mu.L of cationic liposome was added to 500. mu.L of serum-free OPTI-MEM medium, gently mixed, left at room temperature for 5min, the above plasmid and liposome 1:1 were mixed, and left at room temperature for 20min, to obtain a plasmid DNA/liposome complex.

The plasmid DNA/liposome complex was added to a 60mm culture dish at 37 ℃ and 5% CO at 1 mL/dish for 24h ₂ Incubators were incubated for 48 hours for subsequent experiments.

2. Post-transfection RNA extraction

Cells transfected for 48 hours each as described above were collected by digestion, resuspended in 1mL complete medium, 100. mu.L aspirated for RNA extraction, and the remaining resuspension was subjected to subsequent WB sample preparation.

The aspirated 100. mu.L of cell suspension was centrifuged at 4000rpm for 5 minutes, the supernatant was discarded, and 350. mu.L of TRK Lysis Solution (containing 20%. beta. -mercaptoethanol) was added to each sample for Lysis. Each sample was then quenched with 350. mu.L of 70% ethanol (prepared with DEPC water) and mixed by blowing with a gun.

The mixture was transferred to a HiBind RNA Column, centrifuged at 10000g for 1min, and the filtrate was discarded. mu.L of Wash Buffer I was added to each column, 10000g was centrifuged for 1min, and the filtrate was discarded. 500 μ L of Wash Buffer II was added to each column and washed 2 times, each time 10000g was centrifuged for 1min, and the filtrate was discarded. The centrifuge speed was adjusted to the highest speed (17000 g) and centrifuged for 2min to volatilize ethanol from the column. The column was transferred to a clean 1.5mL centrifuge tube without DNA and RNase, left at room temperature for 3-5min, after ethanol was completely evaporated, 50. mu.L of RNase-Free Water was added to each sample, incubated at room temperature for 5min, and centrifuged at 17000g for 1 min. The filtrate was aspirated and added to the column again, incubated at room temperature for 5min, centrifuged at 17000g for 1min to collect RNA, and stored at-80 ℃.

3. RNA reverse transcription, qPCR reaction

The RNA concentration was quantified using a microplate reader (readings were performed using OD 260/280), solutions were prepared according to the number of required PCRs n (n = sample number +1 tube negative control +1 tube positive control), 10. mu.L of reaction system (2. mu.L of 5 XgDNA digaster Buffer, 1. mu.L of gDNA digaster, 100ng of RNA was prepared for each sample, and RNase free ddH was used ₂ O adjusted to 10. mu.L), gently flicked and mixed by a gun, and incubated at 42 ℃ for 2 min. To each sample was added 10. mu.L of 2 XHifair II Supermix plus, and after gently pipetting and mixing with a gun, incubation was performed at 25 ℃ for 5min, 42 ℃ for 30min, and 85 ℃ for 5 min. The collected cDNA was stored at-20 ℃ for further use.

And (3) carrying out reaction on the cDNA product obtained by reverse transcription according to a qPCR kit. The reaction system is as follows: 10 mu L of Hieff qPCR SYBR Green Master Mix (No Rox), 0.4 mu L of each target forward primer and reverse primer, 1 mu L of cDNA template and 20 mu L of sterile ultrapure water complement total volume. And (3) PCR reaction conditions: 95 ℃, 5min, 95 ℃, 10 s, 56 ℃, 30s, 72 ℃, 30s for 40 cycles. Comparing the expression level of the target gene with that of an internal reference and then adopting 2 ^-△△C And (4) calculating by using the method.

And (4) conclusion: as shown in FIG. 5, the new crown pB.1.1.529 mutant strain candidate DNA vaccine can effectively promote the transcription of antigen RNA compared with the empty vector (pVAX 1) 48 hours after in vitro transfection.

Example 4: identification of mammalian cell antigen protein expression of new coronavirus candidate DNA vaccine

To further verify whether the plasmid constructed in example 2 can be efficiently expressed in mammalian cells, it was identified by extracting antigen proteins and Western Blot method.

1. Protein extraction

Transfecting the new crown plasmid pB.1.1.529 into a HEK293T cell strain, removing the transfected culture solution after 48 hours of transfection, washing with precooled PBS once, discarding the PBS, adding 150 mu L of lysate (adding EDTA and protease inhibitor according to a ratio of 1:100 before use), mixing uniformly, and blowing for 10 times. Centrifuge at 4 degrees at 12,000rpm for 5 minutes. The supernatant was aspirated into a 1.5mL centrifuge tube, 50. mu.L of the supernatant was removed for each sample, 12.5. mu.L of 5 XProtein loading buffer was added, and the mixture was boiled in boiling water for 10min and then immediately centrifuged.

2. Sample loading and SDS-PAGE electrophoresis

Adding 62.5 μ L of boiled and centrifuged supernatant sample into SDS-PAGE gel well, switching on power supply, adjusting to constant voltage of 200V, and performing electrophoresis for 45 min. After the electrophoresis, SDS-PAGE was taken out to prepare a membrane. Soaking the PVDF membrane in methanol for 30s for activation, and placing the PVDF membrane in a 1 × rotating membrane equilibrium solution for 1 min.

3. Rotary film

With the positive electrode as the bottom surface, the following steps are carried out: the eBlot L1 membrane-transfer gasket, the PVDF membrane, the gel and the eBlot L1 membrane-transfer gasket were sequentially stacked, and the interlayer air bubbles were removed by a tube every time the stack was stacked. And (3) sealing: the PVDF membrane was removed and placed in a glass box containing 1 XTBST +5% skimmed milk powder and incubated for 1h at room temperature at 90rpm in a shaker. Washing: the PVDF membrane was washed 3 times in 1 XTSST for 10 minutes each time with shaking at 90rpm on a shaker. Primary antibody incubation: PVDF membrane was placed in a primary antibody solution (S-ECD/RBD monoclonal antibody (1), 1ug/mL XG014 antibody was added at 1:2000 dilution) and incubated for 1h at room temperature in a shaker at 90 rpm. Washing: the PVDF membrane was washed 5 times in 1 XTBST for 10 minutes each, shaking at 90rpm on a shaker. And (3) secondary antibody incubation: PVDF membrane was placed in secondary Antibody solution (BD Pharmingen HRP Anti human IgG, 1:5000 dilution and Goat Anti-Rabbit IgG Antibody (H & L) [ HRP ], pAb,1: 4000) for reaction and incubated at room temperature for 1H at 90rpm in a shaker. Washing: the PVDF membrane was washed 5 times in 1 XTSST for 10 minutes each time with shaking at 90rpm on a shaker. Color development: taking 3mL of chemiluminescence solution A and 3mL of chemiluminescence solution B, mixing the chemiluminescence solution A and the chemiluminescence solution B in a ratio of 1: mixing the materials according to the proportion of 1, adding the mixture into a PVDF membrane, incubating for 1-2min, and photographing.

And (4) conclusion: as shown in FIG. 6, the new crown pB.1.1.529 mutant strain candidate DNA vaccine can effectively express antigen protein in cells compared with the empty vector (pVAX 1) 48 hours after transfection in vitro.

Example 5: immunogenicity verification of new crown candidate DNA vaccine

To assess the immunogenicity of the vaccine prepared in example 2, and the impact of the immunization strategy on humoral and cellular immune responses, 6-week-old C57BL/6 female mice, free of specific pathogens, were maintained in an animal facility at the amelanoline Advaccine laboratory (suzhou). Immunization for DNA vaccine: the DNA vaccine described in example 1 was injected into the anterior femoral muscle sequentially according to different grouped injection doses, followed by Electrical Pulses (EP). The Electrical Pulse (EP) device consists of two sets of pulses with a constant current of 0.2 Amp. The second pulse group is delayed by 3 seconds. In each group there are two 52 ms pulses with a delay of 198 ms between the pulses. The first prime was counted as day 0 and the second immunization (boost) was performed on day 14. Grouping experiments: (1) control group vector plasmid pVAX1-25 μ g; (2) the experimental group wild strain pWT-25 μ g; (3) the experimental group mutant pB.1.1.529-25 mug; on day 21, a blood sample was collected from the mouse, and the serum was assayed for the specific antibody titer by ELISA. On day 14 after the booster immunization, immunized mice were sacrificed to analyze cellular immune responses.

1. Evaluation of DNA vaccine elicited antigen-specific humoral immune responses

1.1 ELISA detection of antibody concentration

The RBD protein binding antibodies against the SARS-CoV-2 wild strain and the SARS-CoV-2 B.1.1.529 BA.1 mutant strain were evaluated using ELISA-based methods. Nunc 96-well ELISA plates were coated overnight at 4 ℃ with 1 μ g/mL of SARS-CoV-2 wild strain RBD protein and 1 μ g/mL of SARS-CoV-2 B.1.1.529 BA.1 mutant strain RBD protein (Acro Biosystems, DE, USA), respectively. The plates were washed 3 times and then blocked with 5% Bovine Serum Albumin (BSA) in PBS (0.05% Tween 20, PBST buffer) for 1 hour at 37 ℃. Three serial dilutions of mouse serum were added to each well and incubated for 1 hour at 37 ℃. The plates were washed five times again and then 1: goat anti-mouse IgG-HRP (GenScript, NJ, CN) at 8000 dilution was incubated for 1 hour, followed by detection of bindingAn antibody. After the final wash, the plates were developed by using TMB substrate and 50 μ l/well 2M H ₂ SO ₄ The reaction was terminated. Reading at 450 nm and 620 nm, determining the endpoint of the serum antibody titer as the reciprocal of the highest dilution, wherein the highest dilution of the sample is 2.1 times higher than the absorbance of a negative control (determination standard: experimental group: control (negative) OD450-620 value ≧ 2.1, and the corresponding highest dilution at the OD value is determined as the serum antibody titer).

And (4) conclusion: as shown in FIG. 7, the candidate DNAs of the wild strain pWT and the mutant strain pB.1.1.529 were able to stimulate the experimental animal to produce antigen-specific antibodies significantly at day 7 after the booster immunization. In the above ELISA test, SARS-CoV-2 wild RBD protein and SARS-CoV-2 b.1.1.529 ba.1 mutant RBD protein were used as in vitro envelope antigens, and the results show that the candidate DNA vaccine pWT of new crown wild strain not only can generate antibody against new crown wild antigen, but also can generate antibody against b.1.1.529 mutant antigen, however, the DNA vaccine pb.1.1.529 provided by the present invention also has significant technical effect, and the DNA vaccine pb.1.1.529 provided by the present invention has better effect than candidate DNA vaccine pWT of new crown wild strain, and as mentioned above, pWT is a previous product with excellent immune effect against new crown wild strain, thus better explaining the good immunogenicity and broad spectrum of the vaccine of the present invention.

1.2. Pseudovirus neutralizing antibody detection

Mix 3x10 ⁴ 293-ACE2 cells per well were cultured in 96-well plates in DMEM containing 10% FBS. To detect neutralizing antibody titers, mouse sera (starting from 1:50 dilution) were serially diluted 1:3 in DMEM medium for a total of 6 dilutions. Subsequently, the diluted serum samples were combined with SARS-CoV-2 variant pseudovirus (1X 10) ³ TCID ₅₀ /well) was incubated at 37 ℃ for 1 hour, the mixture was added to 293-ACE2 cells for infection, the cell supernatant was removed after 48 hours of culture, the absolute luciferin emission value in the lysed cells was measured using firefly luciferase assay kit (Promega) and microplate reader, and the results were obtained by mixing the sample with the disease in the same plateThe virus control wells were normalized to calculate relative values. Neutralizing antibody titers were calculated using GraphPad Prism 9 and defined as the reciprocal of the serum dilution (RLU decreased by 50% compared to RLU in virus control wells after subtraction of background RLU in cell control wells).

And (4) conclusion: as shown in FIG. 8, the candidate DNAs of the wild species pWT and pB.1.1.529 mutant strains were able to neutralize viruses such as wild species B.1.1.529 mutant strains at day 7 after the booster immunization. From the result graph, it can be seen that the pb.1.1.529 DNA vaccine provided by the present invention has better effect than the candidate DNA vaccine pWT of new crown wild strain against new crown b.1.1.529 mutant virus, and as mentioned above, pWT is a previous product with excellent immune effect against new crown wild strain, thus better illustrating the good immunogenicity and broad spectrum of the vaccine of the present invention.

2. Further evaluation of the antigen-specific cellular response elicited by DNA vaccines

We investigated whether DNA vaccines could promote cellular immunity by ELISpot analysis. Splenocytes were isolated 14 days after the boost and subjected to IFN-. gamma.ELISpot assay.

2.1 IFN-. gamma.ELISpot assay

On day 14 after boosting, the procedure was performed in a sterile environment, mice were euthanized, spleens were removed, and ground into single cell suspensions; centrifuging to obtain cells, lysing the red blood cell lysate after resuspension, and stopping lysis by PBS containing FBS; filtering, and counting the prepared single cell suspension; single cells were suspended in RPMI1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin. IFN- γ ELISpot assays were performed by using the mouse IFN- γ FlouroPot kit (MabTech, USA). Spleen cell suspensions of each mouse isolated by the above method were cultured at a density of 250,000 to each well coated with anti-IFN-. gamma.antibody, respectively, and CO at 37 deg.C ₂ The culture box is stimulated with SARS-CoV-2 wild strain RBD peptide library and SARS-CoV-2 B.1.1.529 BA.1 mutant strain RBD peptide library for 20 hr. The operation was performed according to the product instructions. Culture medium and PMA/Iono served as negative and positive controls, respectively. Positive spots were quantified by iSpot Reader (AID, Germany). By subtracting negative control wellsSpot Forming Units (SFU) per million cells were calculated.

And (4) conclusion: IFN-gamma ELISPOT results are shown in figure 9, and high-level antigen-specific IFN-gamma response can be effectively induced 14 days after the new crown wild strain pWT and pB.1.1.529 mutant strain candidate DNA vaccine are boosted. In the ELIspot test, the wild RBD protein of neocoronary SARS-CoV-2 and the RBD protein of SARS-CoV-2 b.1.1.529 ba.1 mutant are used as in vitro stimulating peptides, and the conditions are all favorable for wild nucleic acid vaccine pWT of neocoronary, but the mutant DNA vaccine of pb.1.1.529 provided by the present invention also has significant technical effects, and the DNA vaccine of pb.1.1.529 provided by the present invention has better effects than candidate DNA vaccine pWT of new coronary wild strain, and pWT is an early product of excellent immune effect for wild mutant of neocoronary strain, thereby further illustrating the good immunogenicity and broad spectrum of the DNA vaccine of neocoronary pb.1.529 of the present invention.

The invention further detects the immune response capability of the pB.1.1.529 mutant strain candidate DNA vaccine against the new crown Delta (Delta) mutant strain antigen, as shown in figure 10, in an ELIspot test, SARS-CoV-2 Delta mutant strain RBD protein is used as in vitro stimulating peptide, and aiming at the new crown Delta mutant strain antigen, the pB.1.1.529 mutant strain candidate DNA vaccine can also effectively induce high-level antigen specificity IFN-gamma reaction, thereby further illustrating the good immunogenicity and broad spectrum of the new crown pB.1.1.529 mutant strain DNA vaccine.

In conclusion, it can be seen from the results of examples 1-5 that the pB.1.1.529 mutant DNA vaccine of the present invention can be efficiently transcribed and expressed not only in mammalian cells; the pB.1.1.529 mutant strain candidate DNA vaccine can remarkably stimulate experimental animals to generate antigen specific antibodies 7 days after the boost immunity, not only can generate antibodies aiming at the wild-type antigen of the new crown, but also can generate antibodies aiming at the mutant strain antigen of the new crown B.1.1.529; for cellular immune response, the pB.1.1.529 mutant strain candidate DNA vaccine can induce high-level new crown wild type antigen specific IFN-gamma reaction, can also induce high-level new crown B.1.1.529 mutant strain antigen specific IFN-gamma reaction with new crown delta mutant strain antigen specific IFN-gamma reaction, and reflects the good immunogenicity and broad spectrum of the pB.1.1.529 DNA vaccine.

It is worth noting that pWT wild strain vaccine is a product aiming at SARS-CoV-2 wild strain at the early stage of the company, and has entered the clinical stage III at present, and has very excellent immune effect. In the above assays, such as ELISA, pseudovirus neutralization and ELIspot assay, antigen-specific immune response is detected not only using the wild-type RBD protein or virus of Xinguan SARS-CoV-2 as an in vitro envelope antigen or a stimulatory peptide, but also using the RBD protein of the mutant strain of SARS-CoV-2 B.1.1.529 and the RBD protein of the mutant strain of SARS-CoV-2 Deltay as an in vitro envelope antigen or a stimulatory peptide. The pB.1.1.529 DNA vaccine of the invention has obvious better protective effect relative to pWT aiming at the B.1.1.529 mutant strain. And when using new crown SARS-CoV-2 wild type RBD protein as in vitro coating antigen or stimulating peptide, although obviously beneficial to new crown wild type nucleic acid vaccine pWT, the pB.1.1.529 DNA vaccine provided by the invention also obtains significant technical effect, even better than new crown wild type nucleic acid vaccine pWT (especially the result of ELIspot), further proves that the pB.1.1.529 DNA vaccine provided by the invention has better immunogenicity and broad spectrum, and can provide good protective efficacy for SARS-CoV-2 wild type and mutant strains such as B.1.1.529 and delta.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Sequence listing

<110> Amelanchine biopharmaceutical Limited

<120> SARS-CoV-2 virus B.1.1.529 mutant strain DNA vaccine and application

<130> 20220419

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3840

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgtggtggc gcctgtggtg gctgctgctg ctgctgctgc tgctgtggcc catggtgtgg 60

gcctcgcagt gcgtgaacct gaccacacgg acccagctgc ctccagctta cacaaatagc 120

ttcaccagag gcgtgtacta cccggacaag gtgttccggt cctctgtgct gcacagcacc 180

caggacctct tcctgccctt tttcagcaac gtgacctggt tccacgtgat ctctggcaca 240

aacggaacca aaagattcga caaccccgtg ctgcctttca atgatggagt ctacttcgcc 300

tctatcgaaa agagcaacat catccgcggc tggatcttcg gcaccaccct ggacagtaag 360

acccagagcc tgctcatcgt gaacaacgcc acgaacgtgg tgatcaaggt gtgtgaattc 420

caattttgca acgacccctt tctcgaccac aagaacaata aatcttggat ggaaagcgag 480

tttagagtgt acagctctgc taacaactgc actttcgagt acgtgtccca gccattcctg 540

atggacctgg aaggcaagca gggcaatttc aagaacctga gagaattcgt gtttaagaac 600

atcgacggct acttcaaaat ctattctaag cacaccccaa tcatcgtccg ggagcccgag 660

gacctgccac aaggcttcag cgccctggaa cctctggtgg acctgcctat cggaatcaac 720

atcacccggt tccagaccct gctggccctg catcggagct acctgacacc tggcgacagc 780

agctctggct ggaccgccgg cgctgccgca tattacgtcg gctacttgca acctaggacc 840

ttcctgctga aatacaacga gaacggcacc atcacagatg ccgttgattg cgccctggac 900

cccctgagcg aaaccaagtg taccctgaaa tccttcaccg tggaaaaggg catctaccag 960

accagcaact ttagagtaca gcctacagaa tctatcgttc ggtttccaaa cattaccaac 1020

ctgtgtcctt tcgacgaggt gtttaacgcc acacggttcg ccagcgtgta tgcctggaat 1080

agaaagcgga tcagcaactg tgtggccgac tactccgtgc tgtacaatct ggcccccttc 1140

ttcacattta agtgctacgg cgtgtcccct acaaagctga acgacctgtg cttcacaaac 1200

gtgtatgccg atagcttcgt gatccggggc gatgaggtcc ggcagatcgc tcctggccag 1260

acaggcaaca ttgccgacta caactacaag ctgcccgatg acttcaccgg atgtgtgata 1320

gcctggaaca gcaacaagct ggatagcaag gtgagcggca actacaacta cctgtaccga 1380

ctgtttagaa agagcaacct gaaacctttt gagcgggaca tcagcacaga gatctaccaa 1440

gccggcaaca agccttgtaa cggcgtggcc ggcttcaact gttacttccc tctgcggtct 1500

tacagcttcc ggcctacata cggcgtggga caccagccct atagagtggt ggtgctgtca 1560

ttcgagctgc tacatgcccc tgccaccgtg tgcggcccta agaagtctac caacctcgtg 1620

aagaacaagt gcgtgaattt taacttcaat ggactgaagg gcacaggcgt gctgacagag 1680

agcaacaaaa agttcctgcc cttccagcag tttggcagag atatcgctga caccacagac 1740

gccgtgcgcg atcctcagac cctggagatc ctggacatca ccccttgctc ctttggagga 1800

gtgtccgtga tcacacctgg aacgaacacc agcaaccagg ttgccgtgct gtaccagggc 1860

gtgaactgca cagaagttcc tgtggccatc catgccgatc agctgacgcc cacgtggcgg 1920

gtgtactcta ccggcagcaa tgtgttccag accagagccg gctgccttat tggcgctgag 1980

tacgtgaata atagctatga atgcgatatc ccaatcggag ccggcatttg cgccagctac 2040

cagacccaga caaagagtca cagaagagcc agatctgtgg cctcccagag catcatcgca 2100

tataccatga gcctaggagc cgaaaacagc gtcgcctatt ccaacaatag catcgccatc 2160

ccgacaaact tcaccatcag cgtgaccacc gaaatcctgc ccgtgagcat gaccaagaca 2220

agcgtggact gtacaatgta catctgtgga gactccaccg agtgcagcaa cctgctgctg 2280

cagtacggca gcttctgcac ccagctgaag agagccctga cagggatcgc cgtggaacag 2340

gataagaaca cccaagaggt gttcgcccaa gtgaagcaga tctataagac tccacctatt 2400

aagtactttg gcggcttcaa cttcagccaa atcctgcccg atcctagcaa gccaagcaag 2460

cggtccttca tcgaggacct gctgttcaac aaggtgaccc tggccgacgc cggcttcatc 2520

aagcagtatg gcgactgtct gggcgatatc gccgctagag acctgatctg cgcccagaag 2580

ttcaagggcc tgaccgtgct cccacctctg ctcaccgacg agatgatcgc ccagtacacc 2640

tctgccctgc tggccggcac catcaccagc gggtggacat tcggggctgg agctgctctg 2700

caaatcccct tcgccatgca gatggcctac agattcaacg gcatcggcgt tacccagaat 2760

gtgctgtatg aaaaccagaa actgatagct aaccagttca acagcgccat aggcaaaatc 2820

caggatagtc tgagctctac agccagcgcc ctgggaaaac tgcaggatgt ggtgaatcac 2880

aacgcccagg ccctgaatac actggtgaaa caactgagca gcaagttcgg cgccatcagc 2940

agcgtgctga atgatatctt ctctagactg gacccccccg aggccgaggt gcagatcgat 3000

agactgatca ccggcagact gcagtccctg cagacatacg tgactcaaca gctgatcaga 3060

gccgctgaga tcagagcttc tgctaatttg gctgccacaa agatgagcga gtgcgtgctg 3120

ggccagagca aaagagtgga cttctgcggc aagggctacc acctgatgag cttcccccag 3180

agcgcccctc acggcgtcgt gttcctgcac gtgacttacg tgcctgccca agagaagaac 3240

ttcaccaccg cccctgccat ctgccacgac ggcaaggccc acttcccccg ggagggcgtg 3300

ttcgtgagca atggcaccca ctggttcgtg acccaaagaa acttttacga gccccagatt 3360

atcaccaccg acaacacctt cgtgtcaggc aactgcgacg tggtgatcgg catcgtgaac 3420

aacactgtgt acgaccctct gcagcctgag ctggacagct tcaaggagga actggacaag 3480

tacttcaaaa accacacatc tcctgacgtg gacctgggcg atatcagcgg cattaacgcc 3540

tctgtggtga acatccagaa ggaaatcgac agactgaacg aggtggccaa gaacctgaat 3600

gagagcctga tcgacctgca ggagctgggc aagtacgagc agtacatcaa gtggccttgg 3660

tacatctggc tgggctttat cgccggcctg atcgccatcg tgatggtcac catcatgctg 3720

tgctgcatga ccagctgttg cagctgcctg aaaggctgtt gcagctgcgg aagttgctgc 3780

aagtttgacg aggacgactc tgagcctgtg ctgaagggcg tcaagctgca ctacacatga 3840

<210> 2

<211> 3852

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgtggtggc gcctgtggtg gctgctgctg ctgctgctgc tgctgtggcc catggtgtgg 60

gcctctcagt gcgtgaacct gaccaccaga acccagctgc ctcctgctta caccaactcg 120

ttcacacggg gagtgtacta ccccgacaag gtgttcagga gctcagtgct gcatagcacc 180

caagacctgt tcctgccatt cttcagcaac gtcacgtggt tccacgccat ccacgtgtct 240

ggaaccaacg gcaccaagag attcgacaac cccgtgctgc ctttcaacga tggagtgtac 300

ttcgctagca ccgagaagag caacatcatc cggggctgga tcttcggcac cacactggac 360

tccaagacac agagtctgct gatcgtgaac aacgccacca acgtcgtgat caaggtgtgt 420

gagttccagt tctgcaacga tcctttcctc ggcgtttact accacaagaa caacaagagc 480

tggatggaat cagaatttag ggtatattct tctgccaata actgtacgtt tgaatacgtg 540

tctcagcctt tcctaatgga cctggaaggc aaacagggca actttaagaa cctgagagaa 600

ttcgtgttta agaacatcga cggctatttc aagatctaca gtaagcacac ccctatcaac 660

ctggtgcggg acctgcccca ggggttttcc gcccttgaac ctctggtgga cctgcccatt 720

ggcatcaata tcacaagatt ccagaccctg ctggccctgc acagaagcta cctgacccct 780

ggcgacagca gcagcggatg gaccgccggc gccgccgcct actacgtggg atacctgcag 840

cctagaacct tcctactgaa atacaacgaa aacggtacca tcaccgacgc cgtggattgc 900

gctctggacc ctctgagcga aaccaagtgc accctgaaaa gctttaccgt ggagaagggc 960

atttatcaga caagcaactt tcgggtgcag cctaccgaga gcatcgtgag attccctaac 1020

atcaccaacc tgtgtccttt cggcgaggtg ttcaatgcca cacggttcgc cagcgtgtac 1080

gcctggaacc ggaagcggat cagcaactgc gtggccgact acagcgtgct gtataatagc 1140

gccagcttca gcacattcaa gtgctacggc gtgagcccca ccaagctgaa tgatctgtgc 1200

tttaccaacg tgtatgccga tagctttgtg atccgggggg acgaggtaag acagattgcc 1260

ccaggacaga caggcaaaat cgcagattac aactacaaac tgcctgacga cttcaccggc 1320

tgcgttatcg cctggaactc caacaacctg gacagcaagg tgggaggaaa ctacaactac 1380

ctgtaccgac tgttcagaaa gagcaacctg aagccattcg agagagatat ttcgacagag 1440

atctaccagg ccggaagcac accttgcaac ggcgtggaag gcttcaactg ctacttcccc 1500

ctgcagagct acggctttca gcccacaaac ggcgtcggct accagcctta cagagtggtg 1560

gtgctgagct tcgagctgct gcatgcccct gccaccgtgt gcgggcctaa gaagtccaca 1620

aatctggtaa agaataagtg tgtgaacttc aatttcaatg gcctgaccgg aacgggtgtg 1680

ctgaccgaat ctaataagaa gttcctgcct ttccagcagt tcggccgtga tatcgccgac 1740

accaccgacg ctgtccgcga tcctcaaacc ctggaaatcc tggacattac accttgcagc 1800

ttcggcggcg tgtccgtgat cacaccaggc acaaacacca gcaaccaggt ggctgtgctg 1860

taccaggacg tgaactgtac agaggtgcct gtggccatcc acgccgacca gctgacacct 1920

acatggagag tgtattcaac aggcagcaac gtcttccaga ccagagcagg atgcctgatc 1980

ggcgctgagc atgtgaacaa ctcctacgag tgcgacatcc ctatcggcgc cggcatctgc 2040

gctagttacc agactcaaac caactctcct cggcgggcta gaagcgtcgc ctcccagagc 2100

atcatcgctt ataccatgtc tctgggcgcc gagaacagcg tggcctacag caacaactcc 2160

atcgccattc ctaccaactt cacgatctca gttaccaccg agatcctgcc tgtgagcatg 2220

acaaagacca gcgtcgactg caccatgtac atctgcggcg attccacaga atgctccaac 2280

ctgctgctcc agtacggctc tttctgtacc cagctgaaca gagccctgac aggcatcgcc 2340

gtggaacagg ataagaacac tcaggaggtg ttcgcccagg tgaagcagat ctacaagacc 2400

cctccaatca aggactttgg cggctttaat ttcagccaaa tcctcccaga tcctagcaag 2460

cccagcaaga gaagcttcat cgaggacctg ctgttcaaca aggtcaccct ggctgacgcc 2520

ggcttcatca agcagtatgg cgactgcctg ggcgatatcg ccgcgaggga tctaatttgt 2580

gctcagaagt tcaacggcct gaccgtgctg ccccccctgc tgacagacga aatgatcgct 2640

cagtacacat ctgccctgct ggccggcacc atcacgagcg gctggacctt cggagccggc 2700

gccgccctgc agatcccctt cgctatgcag atggcctata gattcaacgg catcggcgtg 2760

acccagaacg tgctgtacga gaaccaaaaa ctgattgcca atcaatttaa ttccgcgatc 2820

ggaaagatcc aggactctct gagctctact gccagcgccc tgggcaagct gcaagacgtg 2880

gtgaaccaga atgctcaagc cctgaacacc ctggtgaagc agctgagcag caatttcgga 2940

gcaatcagct ctgtcctcaa cgacattctg tctagactag acaaggtgga agccgaagtg 3000

cagatcgatc ggcttatcac cggaagactg cagagcctgc agacatatgt tacacagcag 3060

ctgatcagag ccgccgagat cagagccagc gccaacctgg cagccacaaa aatgtccgag 3120

tgcgtcctcg gccaatctaa gcgggttgat ttctgtggca aaggctacca cctgatgagc 3180

ttcccccaaa gcgctcctca cggcgtggtg tttctgcacg tcacctacgt gcccgcccaa 3240

gagaagaact tcaccaccgc ccccgctatc tgccacgacg gcaaggccca cttccctcgg 3300

gaaggcgtgt tcgtgagtaa cggtacacac tggtttgtga cccaaagaaa cttctacgag 3360

cctcagatca tcaccaccga taacaccttt gtgagcggca actgcgatgt ggtgatcggc 3420

atcgtgaaca acacagtata cgaccccctg cagcccgagc tggacagctt taaagaggag 3480

ctcgataagt acttcaagaa ccacacatct ccagacgtgg acctgggcga catcagcggc 3540

atcaacgcca gtgttgtgaa catccagaaa gaaatcgata gactgaacga agtggccaag 3600

aatctgaacg agagcctgat cgacctgcag gagctgggca aatacgagca gtacatcaag 3660

tggccttggt acatctggct gggctttatc gccggcctga tcgccattgt gatggtgaca 3720

atcatgctgt gctgtatgac ctcttgctgc tcctgcctga aaggctgttg tagttgcggc 3780

agctgctgta aattcgatga ggatgactcc gagccggtcc tcaaaggcgt caagctgcac 3840

tacacctgat aa 3852

<210> 3

<211> 3810

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgtttgtgt ttctggtgct gctgccgctg gtgagcagcc agtgcgtgaa cctgaccacc 60

cgcacccagc tgccgccggc gtataccaac agctttaccc gcggcgtgta ttatccggat 120

aaagtgtttc gcagcagcgt gctgcatagc acccaggatc tgtttctgcc gttttttagc 180

aacgtgacct ggtttcatgt gattagcggc accaacggca ccaaacgctt tgataacccg 240

gtgctgccgt ttaacgatgg cgtgtatttt gcgagcattg aaaaaagcaa cattattcgc 300

ggctggattt ttggcaccac cctggatagc aaaacccaga gcctgctgat tgtgaacaac 360

gcgaccaacg tggtgattaa agtgtgcgaa tttcagtttt gcaacgatcc gtttctggat 420

cataaaaaca acaaaagctg gatggaaagc gaatttcgcg tgtatagcag cgcgaacaac 480

tgcacctttg aatatgtgag ccagccgttt ctgatggatc tggaaggcaa acagggcaac 540

tttaaaaacc tgcgcgaatt tgtgtttaaa aacattgatg gctattttaa aatttatagc 600

aaacataccc cgattattgt gcgcgaaccg gaagatctgc cgcagggctt tagcgcgctg 660

gaaccgctgg tggatctgcc gattggcatt aacattaccc gctttcagac cctgctggcg 720

ctgcatcgca gctatctgac cccgggcgat agcagcagcg gctggaccgc gggcgcggcg 780

gcgtattatg tgggctatct gcagccgcgc acctttctgc tgaaatataa cgaaaacggc 840

accattaccg atgcggtgga ttgcgcgctg gatccgctga gcgaaaccaa atgcaccctg 900

aaaagcttta ccgtggaaaa aggcatttat cagaccagca actttcgcgt gcagccgacc 960

gaaagcattg tgcgctttcc gaacattacc aacctgtgcc cgtttgatga agtgtttaac 1020

gcgacccgct ttgcgagcgt gtatgcgtgg aaccgcaaac gcattagcaa ctgcgtggcg 1080

gattatagcg tgctgtataa cctggcgccg ttttttacct ttaaatgcta tggcgtgagc 1140

ccgaccaaac tgaacgatct gtgctttacc aacgtgtatg cggatagctt tgtgattcgc 1200

ggcgatgaag tgcgccagat tgcgccgggc cagaccggca acattgcgga ttataactat 1260

aaactgccgg atgattttac cggctgcgtg attgcgtgga acagcaacaa actggatagc 1320

aaagtgagcg gcaactataa ctatctgtat cgcctgtttc gcaaaagcaa cctgaaaccg 1380

tttgaacgcg atattagcac cgaaatttat caggcgggca acaaaccgtg caacggcgtg 1440

gcgggcttta actgctattt tccgctgcgc agctatagct ttcgcccgac ctatggcgtg 1500

ggccatcagc cgtatcgcgt ggtggtgctg agctttgaac tgctgcatgc gccggcgacc 1560

gtgtgcggcc cgaaaaaaag caccaacctg gtgaaaaaca aatgcgtgaa ctttaacttt 1620

aacggcctga aaggcaccgg cgtgctgacc gaaagcaaca aaaaatttct gccgtttcag 1680

cagtttggcc gcgatattgc ggataccacc gatgcggtgc gcgatccgca gaccctggaa 1740

attctggata ttaccccgtg cagctttggc ggcgtgagcg tgattacccc gggcaccaac 1800

accagcaacc aggtggcggt gctgtatcag ggcgtgaact gcaccgaagt gccggtggcg 1860

attcatgcgg atcagctgac cccgacctgg cgcgtgtata gcaccggcag caacgtgttt 1920

cagacccgcg cgggctgcct gattggcgcg gaatatgtga acaacagcta tgaatgcgat 1980

attccgattg gcgcgggcat ttgcgcgagc tatcagaccc agaccaaaag ccatcgccgc 2040

gcgcgcagcg tggcgagcca gagcattatt gcgtatacca tgagcctggg cgcggaaaac 2100

agcgtggcgt atagcaacaa cagcattgcg attccgacca actttaccat tagcgtgacc 2160

accgaaattc tgccggtgag catgaccaaa accagcgtgg attgcaccat gtatatttgc 2220

ggcgatagca ccgaatgcag caacctgctg ctgcagtatg gcagcttttg cacccagctg 2280

aaacgcgcgc tgaccggcat tgcggtggaa caggataaaa acacccagga agtgtttgcg 2340

caggtgaaac agatttataa aaccccgccg attaaatatt ttggcggctt taactttagc 2400

cagattctgc cggatccgag caaaccgagc aaacgcagct ttattgaaga tctgctgttt 2460

aacaaagtga ccctggcgga tgcgggcttt attaaacagt atggcgattg cctgggcgat 2520

attgcggcgc gcgatctgat ttgcgcgcag aaatttaaag gcctgaccgt gctgccgccg 2580

ctgctgaccg atgaaatgat tgcgcagtat accagcgcgc tgctggcggg caccattacc 2640

agcggctgga cctttggcgc gggcgcggcg ctgcagattc cgtttgcgat gcagatggcg 2700

tatcgcttta acggcattgg cgtgacccag aacgtgctgt atgaaaacca gaaactgatt 2760

gcgaaccagt ttaacagcgc gattggcaaa attcaggata gcctgagcag caccgcgagc 2820

gcgctgggca aactgcagga tgtggtgaac cataacgcgc aggcgctgaa caccctggtg 2880

aaacagctga gcagcaaatt tggcgcgatt agcagcgtgc tgaacgatat ttttagccgc 2940

ctggataaag tggaagcgga agtgcagatt gatcgcctga ttaccggccg cctgcagagc 3000

ctgcagacct atgtgaccca gcagctgatt cgcgcggcgg aaattcgcgc gagcgcgaac 3060

ctggcggcga ccaaaatgag cgaatgcgtg ctgggccaga gcaaacgcgt ggatttttgc 3120

ggcaaaggct atcatctgat gagctttccg cagagcgcgc cgcatggcgt ggtgtttctg 3180

catgtgacct atgtgccggc gcaggaaaaa aactttacca ccgcgccggc gatttgccat 3240

gatggcaaag cgcattttcc gcgcgaaggc gtgtttgtga gcaacggcac ccattggttt 3300

gtgacccagc gcaactttta tgaaccgcag attattacca ccgataacac ctttgtgagc 3360

ggcaactgcg atgtggtgat tggcattgtg aacaacaccg tgtatgatcc gctgcagccg 3420

gaactggata gctttaaaga agaactggat aaatatttta aaaaccatac cagcccggat 3480

gtggatctgg gcgatattag cggcattaac gcgagcgtgg tgaacattca gaaagaaatt 3540

gatcgcctga acgaagtggc gaaaaacctg aacgaaagcc tgattgatct gcaggaactg 3600

ggcaaatatg aacagtatat taaatggccg tggtatattt ggctgggctt tattgcgggc 3660

ctgattgcga ttgtgatggt gaccattatg ctgtgctgca tgaccagctg ctgcagctgc 3720

ctgaaaggct gctgcagctg cggcagctgc tgcaaatttg atgaagatga tagcgaaccg 3780

gtgctgaaag gcgtgaaact gcattatacc 3810

Claims

1. A DNA molecule, wherein the nucleotide sequence of said DNA molecule is as set forth in SEQ ID NO: 1 is shown.

2. A biomaterial, characterized in that it comprises at least one of (a) - (b):

(a) a recombinant expression vector comprising the DNA molecule of claim 1;

(b) a host cell comprising the DNA molecule of claim 1 or the recombinant expression vector of (a).

3. The biomaterial according to claim 2, wherein the recombinant expression vector is a eukaryotic expression vector having a vector backbone of pVAX 1.

4. Use of the DNA molecule of claim 1 or the biomaterial of claim 2 or 3 in (a) or (B) as follows:

(A) preparing vaccine for preventing SARS-CoV-2 virus infection;

(B) preparing a medicament for preventing related diseases caused by SARS-CoV-2 virus;

the SARS-CoV-2 virus includes mutant strain B.1.1.529, wild strain or mutant strain B.1.617.2.

5. A DNA vaccine comprising the DNA molecule of claim 1 or the recombinant expression vector of claim 2 or 3.

6. The DNA vaccine of claim 5, further comprising a pharmaceutically acceptable adjuvant, carrier, diluent or excipient.

7. The DNA vaccine of claim 6, wherein the adjuvant comprises a TLRs ligand and/or a metal ion and/or a cytokine adjuvant.

8. The method for producing a DNA vaccine according to any one of claims 5 to 7, wherein a recombinant vector comprising the DNA molecule according to claim 1 is introduced into a host cell and cultured, and the recombinant vector in the host cell is extracted to obtain the DNA vaccine.