CN113528549B - DNA molecule for encoding novel coronavirus B.1.351 mutant strain antigen, DNA vaccine and application - Google Patents

Abstract

The invention relates to the technical field of biology, and particularly provides a DNA molecule for encoding a novel coronavirus B.1.351 mutant strain antigen, a DNA vaccine and application. The nucleic acid sequence SEQ ID NO.1 provided by the invention can be efficiently transcribed and expressed in a eukaryotic expression system, has immunogenicity as shown in humoral immunity and cellular immune response, and the nucleic acid vaccine taking the nucleic acid sequence as an active ingredient also has good immunogenicity.

Description

DNA molecule for encoding novel coronavirus B.1.351 mutant strain antigen, DNA vaccine and application

Technical Field

The invention relates to the technical field of biology, in particular to a DNA molecule for coding a novel coronavirus B.1.351 mutant strain antigen, a DNA vaccine and application.

Background

SARS-CoV-2 is a single-stranded positive-strand RNA virus with an envelope structure, which is highly susceptible to mutation. At present, according to the new coronavirus pedigree information published by the GISAID database, more than 700 mutations occur globally, wherein the transmission capability is strong, and the widely distributed mutant strains are 6 in total, namely: b.1.1.7 mutant, B.1.351 mutant, P.1 mutant, B.1.2 mutant, B.1 mutant, B.1.525 mutant and B.1.617 mutant. Among them, the B.1.351 mutant has 10 defined mutations on the S protein. The research shows that the E484K mutation can obviously improve the capability of the live virus and the pseudovirus to resist monoclonal neutralizing antibodies and vaccine serum. In conclusion, there is an urgent need to develop more effective vaccines against mutant strains.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

It is an object of the present invention to provide a DNA molecule encoding a novel coronavirus B.1.351 mutant antigen, which at least alleviates 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 invention also aims to provide application of the biological material.

The fourth purpose of the invention is to provide a novel coronavirus B.1.351 mutant strain DNA vaccine comprising 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 encoding a novel coronavirus B.1.351 mutant strain antigen, wherein the DNA molecule has a nucleotide sequence shown as SEQ ID NO.1 or a nucleotide sequence which has at least 90% of identity with the nucleotide sequence shown as SEQ ID NO. 1.

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 cell line, DH5 alpha, Top10, BL21, DH10B and the like cells.

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 the prevention and/or treatment of a novel coronavirus infection;

(B) preparing a medicament for preventing and/or treating related diseases caused by the novel coronavirus.

Further, the novel coronavirus comprises a B.1.351 mutant strain, a wild strain, a B.1.1.7 mutant strain, a P.1 mutant strain, a B.1.2 mutant strain, a B.1 mutant strain, a B.1.525 mutant strain or a B.1.617 mutant strain.

The invention also provides a novel coronavirus B.1.351 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 the novel coronavirus.

Preferably, the adjuvant comprises an aluminium adjuvant and/or a TLRs ligand and/or a metal ion such as Mn2⁺、Zn2⁺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) against infection by a novel coronavirus;

(iii) prevention of immunopathological damage;

the novel coronavirus comprises a B.1.351 mutant strain, a wild strain, a B.1.1.7 mutant strain, a P.1 mutant strain, a B.1.2 mutant strain, a B.1 mutant strain, a B.1.525 mutant strain or a B.1.617 mutant strain.

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

the invention optimizes the coding DNA sequence of the novel coronavirus B.1.351 mutant Spike protein by using different optimization algorithms to obtain the DNA molecule with the nucleotide sequence shown in SEQ ID NO.1 or the nucleotide sequence which is at least 90 percent identical to the nucleotide sequence shown in SEQ ID NO. 1. The DNA molecule can efficiently transcribe and express the novel coronavirus B.1.351 mutant Spike antigen, has immunogenicity, and can induce specific humoral immunity and cellular immune response.

Based on the beneficial effects of the DNA molecule encoding the novel coronavirus B.1.351 mutant strain antigen, the invention also provides a DNA vaccine comprising the DNA molecule. The DNA vaccine can be effectively transcribed and expressed in mammalian cells, has good immunogenicity, can remarkably stimulate experimental animals to generate antigen specific antibodies on 14 days after primary immunization and 7 days after boosting immunization for humoral immune response, and has good neutralizing activity on wild viruses, B.1.351, P.1, B.1.617 and other viruses; for cellular immune response, the DNA vaccine can not only induce high-level antigen-specific IFN-gamma response, generation of antigen-specific CD4TNF alpha T cell subsets and CD8IFN gamma T cell subsets, but also induce high-activity antigen-specific CTL response.

Based on the above, the DNA vaccine provided by the invention can adjust the immune function of the organism, effectively prevent the infection of the novel coronavirus and the mutant strain thereof, especially the B.1.351 mutant strain, and simultaneously can treat diseases caused by the novel coronavirus and the mutant strain thereof, especially the B.1.351 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 shows the qPCR results of the expression of the candidate DNA vaccines of the new crown wild strain and the B.1.351 mutant strain provided by the embodiment of the invention;

FIG. 2 shows the results of antigen protein expression of candidate DNA vaccines of the Xinguan wild strain and the B.1.351 mutant strain provided by the embodiment of the present invention;

FIG. 3 shows the results of antigen-specific antibodies 14 days after the initial immunization of the candidate DNA vaccines of the wild strain and the mutant strain B.1.351 of the Xinguan provided in the embodiment of the present invention;

FIG. 4 shows the results of antigen-specific antibodies at day 7 after the boosting immunization of the candidate DNA vaccines of the wild strain and the mutant strain B.1.351 of the Xinguan provided in the embodiment of the present invention;

FIG. 5 shows the neutralizing antibody results of the candidate DNA vaccine of the new crown wild strain and the B.1.351 mutant strain provided by the embodiment of the invention on the 7 th day after the boosting immunization;

FIG. 6 shows the result of antigen-specific ELISOPT at day 14 after the primary immunization of the candidate DNA vaccine of the new crown wild strain and the B.1.351 mutant strain provided by the embodiment of the invention;

FIG. 7 shows the results of antigen-specific ELISOPT at day 7 after the boosting immunization of the candidate DNA vaccines of the new crown wild strain and the B.1.351 mutant strain provided by the embodiment of the invention;

FIG. 8 shows the result of antigen-specific CD4TNF α T cell subsets at day 7 after the boosting immunization of the candidate DNA vaccines of the wild strain and the mutant strain B.1.351 provided by the embodiment of the invention;

FIG. 9 shows the result of antigen-specific CD8TFN alpha T cell subsets at day 7 after the booster immunization of the candidate DNA vaccines of the new crown wild strain and the B.1.351 mutant strain provided by the embodiment of the invention;

FIG. 10 shows the result of antigen-specific CD8IFN γ T cell subsets at day 7 after boosting immunization with candidate DNA vaccines of the new crown wild strain and B.1.351 mutant strains provided by the embodiment of the present invention;

FIG. 11 shows the result of antigen-specific in vivo CTL 14 days after the initial immunization of the candidate DNA vaccines of the wild strain and the mutant strain B.1.351 provided in the example of the present invention;

FIG. 12 shows the antigen-specific in vivo CTL results at day 7 after the booster immunization of the candidate DNA vaccines of the new crown wild strain and the B.1.351 mutant strain provided by the embodiment of the invention.

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 DNA sequence encoding the Spike protein of the novel coronavirus B.1.351 mutant strain is optimized by different optimization algorithms to obtain a DNA molecule with the nucleotide sequence shown in SEQ ID NO.1 or a nucleotide sequence at least 90% identical to the nucleotide sequence shown in SEQ ID NO. 1. The DNA molecule can be transcribed efficiently, is more beneficial to efficiently expressing the novel coronavirus B.1.351 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 having at least 90% (e.g., may be, but is not limited to, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to the nucleotide sequence represented by SEQ ID No.1 described in the present invention.

Optionally, the optimization also comprises replacing a signal peptide of the novel coronavirus B.1.351 mutant strain gene with a high-expression signal peptide so as to improve the expression efficiency of the DNA sequence in a 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 may be a eukaryotic expression vector which may be transformed into a target cell by integration into the genome of the cell, and the protein encoded by the DNA molecule is produced by cellular transcription and translation machinery. 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 insertion of transcription termination sequences and PTIS. 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. Wherein the host cell may be a prokaryotic cell, such as a bacterial cell, typically E.coli: (E.coli) Or eukaryotic cells, which typically may be insect, yeast, avian, or mammalian cells, as well as other suitable host cells. The cell comprises HEK293, CHO, COS-7 cell strain, DH5 alpha, Top10, BL21. DH10B 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-mentioned DNA molecule or biomaterial for the preparation of a vaccine for the prevention and/or treatment of infection by the novel coronavirus and/or for the preparation of a medicament for the prevention and/or treatment of a disease associated with the novel coronavirus, such as lung injury, brain injury, liver and kidney injury, and heart injury.

Preferably, the novel coronavirus comprises a b.1.351 mutant strain, a wild strain, a b.1.1.7 mutant strain, a p.1 mutant strain, a b.1.2 mutant strain, a b.1 mutant strain, a b.1.525 mutant strain, or a b.1.617 mutant strain.

Based on the beneficial effects of the DNA molecule encoding the novel coronavirus B.1.351 mutant strain antigen, the invention also provides a DNA vaccine comprising the DNA molecule.

The DNA vaccine can effectively transcribe and express a novel coronavirus B.1.351 mutant Spike antigen in a mammalian cell, excites a more efficient immune response, can remarkably excite an experimental animal to generate an antigen specific antibody on both 14 th day after primary immunization and 7 th day after boosting immunization for humoral immune response, and has better neutralizing activity for wild viruses, B.1.351 viruses, P.1 viruses, B.1.617 viruses and the like; for cellular immune response, the DNA vaccine can not only induce high-level antigen-specific IFN-gamma response, generation of antigen-specific CD4TNF alpha T cell subsets and CD8IFN gamma T cell subsets, but also induce high-activity antigen-specific CTL response.

In some embodiments, the DNA vaccine further comprises 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. Wherein is pharmaceutically acceptableThe adjuvant may be selected from aluminium adjuvants and/or TLRs ligands and/or metal ions such as Mn2⁺、Zn2⁺And/or cytokine and/or chemokine adjuvants, and the like.

In other embodiments, the DNA vaccine further comprises at least one therapeutic agent for the novel coronavirus to enhance the therapeutic effect of the vaccine on the disease associated with the novel coronavirus.

The action mechanism of the DNA vaccine provided by the invention is as follows: the coding DNA of the surface antigen Spike antigen of the novel coronavirus B.1.351 mutant strain is optimized by different optimization algorithms, the wild type gene signal peptide is replaced by a high-efficiency expression signal peptide and then inserted into a eukaryotic expression vector, the eukaryotic expression vector is introduced into a host cell, so that the virus Spike antigen is efficiently expressed in the host cell, and the antiviral humoral immune response and the cellular immune response are systematically activated through the antigen presentation process. 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) against infection by a novel coronavirus;

(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: construction process of DNA vaccine

1. Preparation method of new coronavirus candidate DNA vaccine

1.1. Construction of plasmids

According to the B.1.351 mutant strain sequence (EPI _ ISL _860630, GISAID), the nucleotide sequence shown in SEQ ID NO.1 is obtained by combining empirical optimization, and the nucleotide sequence shown in SEQ ID NO.1 is inserted between the BanH I and Xho I sites of the pVAX1 vector to obtain the new coronavirus B.1.351 mutant strain plasmid (pB.1.351).

The nucleotide sequence shown in SEQ ID NO.2 is obtained through optimization according to a new crown wild type sequence (MN 908947.3, NCBI), and the nucleotide sequence shown in SEQ ID NO.2 is inserted between the BanH I and Xho I sites of a pVAX1 vector to obtain a new crown virus 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 freezer at-80 ℃ 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 h. Plasmid extraction was performed with an EndoFreen Plasmid Maxi kit (QIAGEN, Germany): centrifuging the cultured bacterial liquid for 12-16h at 8000rpm and 4 ℃ for 10min, removing the supernatant, collecting the bacterial body, adding 10ml of Buffer P1 heavy suspension, adding 10ml of Buffer P2, slightly reversing for 4-6 times, mixing, incubating at room temperature for 5min, and fully lysing. 10ml of Buffer P3 was added to the mixture, after termination of lysis by gentle inversion for 4-6 times, all were transferred to a QIAfilter Cartridge, incubated at room temperature for 10min, and the supernatant was filtered by adding a 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. Each tube was precipitated with 10.5ml 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 μ l of endotoxin-free water into each sample to resuspend the plasmid, thereby obtaining the DNA vaccine plasmid.

Example 2: mammalian cell transcriptional identification of new coronavirus candidate DNA vaccines

To verify whether the plasmid constructed in example 1 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 DMEM medium containing 10% calf serum₂Culturing 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 10⁶The density of cells/well was plated on a 60mm dish and 5ml growth medium (without 1% double antibody) was added at 37 ℃ with 5% CO₂Culturing in an incubator for 24 h.

Mu.g of sterile plasmid (pWT) of the wild species of the New crown and 4. mu.g of sterile plasmid (pB.1.351) of the mutant B.1.351 were added to 500. mu.l of serum-free OPTI-MEM medium, and gently mixed, and 24. mu.l of cationic liposome was added to 500. mu.l of serum-free OPTI-MEM medium, and gently mixed, and left at room temperature for 5min, and the two plasmids pWT and pB.1.351 were mixed with liposome 1:1, respectively, 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 1 ml/dish and incubated for 24 hours at 37 ℃ with 5% CO₂The incubators were incubated for 24 hours (24H), 48 hours (48H), and 72 hours (72H), respectively, for subsequent experiments.

2. Post-transfection RNA extraction

The cells transfected to 24 hours, 48 hours, and 72 hours above were collected by digestion, resuspended in 1ml complete medium, 100. mu.l was 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 an additional 350. mu.l of 70% ethanol (made up 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. mu.l of Wash Buffer II was added to each column and washed 2 times, each time, 10000g was used for centrifugation 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 ℃.

RNA reverse transcription, qPCR reaction

The RNA concentration was quantified using a microplate reader (readings were performed using OD 260/280), a solution was prepared based on the number of desired PCR samples n (n = sample number +1 tube negative control +1 tube positive control), and 10. mu.l of a reaction system (2. mu.l of 5 Xg DNA digaster Buffer, 1. mu.l of gDNA digaster, 100ng of RNA) was prepared for each sample, and RNase free ddH was used₂The volume was adjusted to 10. mu.l) by O, the mixture was gently blown down by a gun and incubated at 42 ℃ for 2 min. Mu.l of 2 XHifair II Supermix plus was added to each sample, 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 mul of Hieff qPCR SYBR Green Master Mix (No Rox), 0.4 mul of target forward primer and target reverse primer respectively, 1 mul of cDNA template and sterile ultrapure water for complementing the total volume20 μ l. 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^-△△CAnd (4) calculating by using the method.

And (4) conclusion: as shown in FIG. 1, the empty vector (pVAX 1) can promote the transcription of antigen RNA at high level after 24 hours, 48 hours and 72 hours of in vitro transfection, and the transcription level is the highest 24 hours after transfection, for the new crown wild strain and the candidate DNA vaccine of B.1.351 mutant strain.

Example 3: identification of mammalian cell antigen protein expression of new coronavirus candidate DNA vaccine

To further verify whether the plasmid constructed in example 1 can be efficiently expressed in mammalian cells, it was identified by extracting antigen proteins and Western Blot method.

1. Protein extraction

Transfecting a new crown wild strain plasmid (pWT) and a B.1.351 mutant strain plasmid (pB.1.351) into a HEK293T cell strain, removing a transfected culture solution after 48 hours of transfection, washing once by using precooled PBS (PBS), discarding the PBS, adding 150 mu l of lysate (adding EDTA and protease inhibitor according to a ratio of 1:100 before use), uniformly mixing, and then 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 XP 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: the PVDF membrane was reacted with a primary antibody (S-ECD/RBD monoclonal antibody (1), 1:2000 dilution) and incubated at 90rpm in a shaker at room temperature for 1 hour. Washing: the PVDF membrane was washed 5 times in 1 XTSST for 10 minutes each time, shaking at 90rpm in a shaker. And (3) secondary antibody incubation: the PVDF membrane was placed in a secondary antibody solution (BD Pharmingen HRP Anti human IgG, 1:5000 dilution) for reaction and incubated at room temperature for 1h at 90rpm on 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 proportion 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. 2, the candidate DNA vaccines of the new crown wild strain and b.1.351 mutant strain were able to express the antigen Spike protein at a high level in the cell compared to the empty vector (pVAX 1) 48 hours after in vitro transfection.

Example 4: immunogenicity verification of new crown candidate DNA vaccine

To assess the immunogenicity of the vaccine prepared in example 1, 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 purchased from Calvens bagger and maintained in the animal facility at the Amelanchivenn Advaccine laboratory (Suzhou). For vaccination with DNA vaccines: 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) experimental group B.1.351 mutant pB.1.351-2.5 μ g; (4) experimental group B.1.351 mutant pB.1.351-25 μ g; on day 14, 21, a blood sample was collected from the mouse, and the serum was assayed for the specific antibody titer by ELISA. Immunized mice were sacrificed at day 14 post-primary and day 7 post-booster immunizations to analyze cellular immune responses.

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

1.1 ELISA detection of antibody concentration

Antibody binding to SARS-CoV-2 RBD protein was assessed using an ELISA-based method. Nunc 96 well ELISA plates were coated overnight at 4 ℃ with 1 μ g/mL SARS-Cov-2 RBD protein (Acro Biosystems, DE, USA). 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 dilutions was incubated for 1 hour, followed by detection of bound antibody. After the final wash, the plates were developed by using TMB substrate and 50 μ l/well 2M H₂SO₄The 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. 3 and 4, the 14 th day after the primary immunization and the 7 th day after the booster immunization of the new crown b.1.351 mutant strain and the wild strain candidate DNA vaccine can remarkably stimulate the experimental animal to produce antigen-specific antibodies. In the above ELISA test, the neocoronary wild-type SARS-Cov-2 RBD protein was used as the in vitro envelope antigen, and the above conditions are all favorable for the neocoronary wild-type nucleic acid vaccine pWT, however, the b.1.351 mutant DNA vaccine provided by the present invention has even better technical effect, and as mentioned above, pWT is a prior product with excellent immune effect, which further illustrates the good immunogenicity and broad spectrum of the vaccine of the present invention.

1.2. Pseudovirus neutralizing antibody detection

Will be 1 × 10⁴Huh-7 cells per well were seeded in 96-well plates in DMEM containing 10% FBS. Before infection will be connectedThe cells of the species were cultured for eight hours. To detect neutralizing antibody titers, mouse sera (starting from 1:40 dilution) were serially diluted 1:2 in DMEM medium for a total of nine dilutions. 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 further incubation for 12 hours, the supernatant was replaced with fresh DMEM medium (containing 2% FBS). After another 48 hours of culture, the cell supernatant was removed, and the absolute luciferin luminescence value in the lysed cells was measured using a firefly luciferase assay kit (Promega) and a microplate reader, and the relative value was calculated by normalizing with the virus control well in the same plate. 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: the results are shown in fig. 5, the candidate DNA vaccine of the new corona b.1.351 mutant strain can have better neutralizing activity for wild-type new corona virus, b.1.351 mutant strain, p.1 mutant strain, b.1.617 mutant strain and other viruses at 7 days after the booster immunization, which shows that the candidate DNA vaccine of the b.1.351 mutant strain of the present application has good broad spectrum protection potential.

2. Further evaluation of DNA vaccine elicited antigen-specific cellular responses

A polypeptide library (S peptide) of the S antigen specific epitope peptide is predicted and synthesized, and the peptide library is used for stimulating the splenocytes of the mice after vaccine immunization.

2.1. Immune cell specificity stimulation detection of cellular immune response

We investigated whether DNA vaccines could promote cellular immunity by ELISpot analysis. Splenocytes were isolated 14 days after the primary immunization and 7 days after the booster immunization, respectively, and subjected to IFN-. gamma.positive cell ELISpot experiments.

2.2 isolation of splenocytes

On day 14 after primary immunization and 7 after booster immunization, mice were euthanized in a sterile environment, 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.

2.3 IFN-. gamma.ELISpot assay

IFN- γ ELISpot assays were performed by using the mouse IFN- γ ELISpot kit (Dakewe, SZ, China). Spleen cell suspension of each mouse isolated by the above method was inoculated at a density of 250,000 to each well coated with anti-IFN-. gamma.antibody, and CO at 37 deg.C₂The incubators were stimulated with SARS-CoV-2 RBD peptide library for 20 hours at a peptide library concentration of 10. mu.g/mL (final concentration) per well (in RPMI + 10% FBS). 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, Stra beta berg, Germany). Spot Forming Units (SFU) per million cells were calculated by subtracting negative control wells.

And (4) conclusion: IFN-gamma ELISPOT results are shown in FIGS. 6-7, and the new crown B.1.351 mutant strain and the wild strain candidate DNA vaccine can effectively induce high-level antigen-specific IFN-gamma reaction on 14 days after primary immunization and 7 days after booster immunization. In the ELIspot test, the neocorolla wild-type SARS-Cov-2 RBD protein is used as in vitro stimulating peptide, and the conditions are all favorable for the neocorolla wild-type nucleic acid vaccine pWT, but the b.1.351 mutant DNA vaccine provided by the invention also has a remarkable technical effect, and even has a better technical effect on the 14 th day after the initial immunization, as mentioned above, pWT is a previous product with an excellent immune effect, and better immunogenicity and broad spectrum of the neocorolla b.1.351 mutant DNA vaccine are demonstrated.

3. Further evaluation of the effects of the antigen-specific cellular immune response elicited by the vaccine, in particular the effects of CD4 and CD8T cell function, splenocytes were isolated 7 days after booster immunization and subjected to flow cytometry assays.

Isolation of splenocytes: 7 days after the booster immunization, the procedure was carried out in a sterile environment, the mice were euthanized, the spleens were taken out, 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.

Flow cytometry detection experiment: spleen cell suspension from each mouse obtained by the above method, 37 ℃, 5% CO₂Next, the cells were stimulated with SARS-CoV-2 RBD peptide library or PMA/Iono, while blocking with 1. mu.g/ml Breededlin A (BD, CA, USA) for 6 hours. Extracellular and intracellular cytokine staining of splenocytes, stimulated splenocytes were stained with FVD-eFluor780, then washed, and stained with anti-mouse CD4, CD8a antibody, respectively, in the dark at room temperature for 30 minutes. Cells were permeabilized with the fixation/permeation buffer and stained intracellularly with anti-mouse IFN-. gamma.and anti-mouse TNF-. alpha.for 45 minutes at 4 ℃. Cells were washed twice and resuspended in 200 μ L PBS before being harvested using a flow cytometer (ThermoFisher, MA, usa) and analyzed using FlowJo software (BD, CA, usa).

And (4) conclusion: the results are shown in fig. 8-10, and the new crown b.1.351 mutant strain and wild strain candidate DNA vaccine can significantly induce the generation of antigen-specific CD4TNFa T cell subset and CD8IFN γ T cell subset at day 7 after the boost. In the FACS test, the wild SARS-Cov-2 RBD protein of neoguan is used as the in vitro stimulating peptide, which is beneficial to the wild-type nucleic acid vaccine pWT of neoguan, however, the mutant b.1.351 DNA vaccine provided by the present invention also achieves significant technical effects, as mentioned above, pWT is a prior product with excellent immune effect, which further illustrates the excellent immunogenicity and broad spectrum of the mutant b.1.351 DNA vaccine of the present invention.

4. Immune cell specific stimulation detection of in vivo CTL response

Since CTL responses play a key role in combating viral infection and eliminating virus-infected cells, in vivo CTL assays were performed at day 14 after the primary immunization and at day 7 after the booster immunization in order to explore the effect of example 1 on cytotoxic T cell function.

Spleen cells of blank C57BL/6 mice (1.5X 10)⁸) Incubating with 10. mu.g of S peptide pool, and collectingBlank C57BL/6 mouse spleen cells (1.5X 10)⁸) The polypeptides were not incubated. 5% CO at 37 ℃₂And culturing for 4 h. Cells were labeled with eflour450 and the polypeptide incubated for groups (1X 10)⁷Cell/ml + 5. mu.M, high-stained), control group incubated without polypeptide (1X 10)⁷Cells/ml +0.5 μ M, low staining). Mixing high-staining cells and low-staining cells at a ratio of 1:1 to obtain a final total cell concentration of 2 × 10⁷Cells/ml. By tail vein injection, 4X 10⁶Injecting the mixed cells into an immune group mouse, taking spleen cells after 4 hours, performing flow-type machine, and collecting a sample. The in vivo killing rate is calculated as follows.

Wherein T represents a Targets group and NT represents a Non Targets group.

And (4) conclusion: as shown in FIGS. 11-12, the new crown B.1.351 mutant strain and the wild strain candidate DNA vaccine were able to induce a significant high activity of antigen-specific CTL response both at day 14 after the initial immunization and at day 7 after the booster immunization.

In conclusion, it can be seen from the results of examples 1-4 that the B.1.351 mutant DNA vaccine of the present invention can be efficiently transcribed and expressed in mammalian cells; the B.1.351 mutant strain candidate DNA vaccine can obviously stimulate experimental animals to generate antigen-specific antibodies on the 14 th day after primary immunization and the 7 th day after boosting immunization for humoral immune response, and has better neutralizing activity for wild viruses, B.1.351 viruses, P.1 viruses, B.1.617 viruses and the like; for cellular immune response, the B.1.351 mutant strain candidate DNA vaccine can not only induce high-level antigen-specific IFN-gamma response, generation of antigen-specific CD4TNFa T cell subset and CD8IFN gamma T cell subset, but also induce high-activity antigen-specific CTL response.

It is worth noting that the pWT wild strain vaccine is a product aiming at wild strains at the early stage of the company, is about to enter the phase III clinic at present, and has very excellent immune effect. In the above tests, for example, in the detection of ELISA, ELIspot and FACS, the Xinguan wild type SARS-Cov-2 RBD protein is used as in vitro envelope antigen or stimulating peptide, and the above conditions are all favorable for Xinguan wild type nucleic acid vaccine pWT, however, the B.1.351 mutant strain DNA vaccine provided by the invention also obtains significant, even better technical effects, and better shows the good immunogenicity and broad spectrum of the B.1.351 mutant strain DNA vaccine.

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> DNA molecule for encoding novel coronavirus B.1.351 mutant strain antigen, DNA vaccine and application

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 3840

<212> DNA

<213> 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 tccacgctat ccacgtgtct 240

ggcacaaacg gaaccaaaag attcgctaac cccgtgctgc ctttcaatga tggagtctac 300

ttcgcctcta ccgaaaagag caacatcatc cgcggctgga tcttcggcac caccctggac 360

agtaagaccc agagcctgct catcgtgaac aacgccacga acgtggtgat caaggtgtgt 420

gaattccaat tttgcaacga cccctttctc ggcgtgtact accacaagaa caataaatct 480

tggatggaaa gcgagtttag agtgtacagc tctgctaaca actgcacttt cgagtacgtg 540

tcccagccat tcctgatgga cctggaaggc aagcagggca atttcaagaa cctgagagaa 600

ttcgtgttta agaacatcga cggctacttc aaaatctatt ctaagcacac cccaatcaac 660

ctggtccggg gcctgccaca aggcttcagc gccctggaac ctctggtgga cctgcctatc 720

ggaatcaaca tcacccggtt ccagaccctg catatcagct 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 tcggcgaggt gtttaacgcc acacggttcg ccagcgtgta tgcctggaat 1080

agaaagcgga tcagcaactg tgtggccgac tactccgtgc tgtacaatag cgccagcttc 1140

tctacattta agtgctacgg cgtgtcccct acaaagctga acgacctgtg cttcacaaac 1200

gtgtatgccg atagcttcgt gatccggggc gatgaggtcc ggcagatcgc tcctggccag 1260

acaggcaaca ttgccgacta caactacaag ctgcccgatg acttcaccgg atgtgtgata 1320

gcctggaaca gcaacaacct ggatagcaag gtgggcggca actacaacta cctgtaccga 1380

ctgtttagaa agagcaacct gaaacctttt gagcgggaca tcagcacaga gatctaccaa 1440

gccggctcta ccccttgtaa cggcgtgaag ggcttcaact gttacttccc tctgcagtct 1500

tacggattcc agcctacata cggcgtggga taccagccct atagagtggt ggtgctgtca 1560

ttcgagctgc tacatgcccc tgccaccgtg tgcggcccta agaagtctac caacctcgtg 1620

aagaacaagt gcgtgaattt taacttcaat ggactgacag 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

cacgtgaata atagctatga atgcgatatc ccaatcggag ccggcatttg cgccagctac 2040

cagacccaga caaatagtcc tagaagagcc agatctgtgg cctcccagag catcatcgca 2100

tataccatga gcctaggagt ggaaaacagc gtcgcctatt ccaacaatag catcgccatc 2160

ccgacaaact tcaccatcag cgtgaccacc gaaatcctgc ccgtgagcat gaccaagaca 2220

agcgtggact gtacaatgta catctgtgga gactccaccg agtgcagcaa cctgctgctg 2280

cagtacggca gcttctgcac ccagctgaac agagccctga cagggatcgc cgtggaacag 2340

gataagaaca cccaagaggt gttcgcccaa gtgaagcaga tctataagac tccacctatt 2400

aaggactttg gcggcttcaa cttcagccaa atcctgcccg atcctagcaa gccaagcaag 2460

cggtccttca tcgaggacct gctgttcaac aaggtgaccc tggccgacgc cggcttcatc 2520

aagcagtatg gcgactgtct gggcgatatc gccgctagag acctgatctg cgcccagaag 2580

ttcaatggcc 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 ggtgaatcag 2880

aacgcccagg ccctgaatac actggtgaaa caactgagca gcaatttcgg cgccatcagc 2940

agcgtgctga atgatatcct gtctagactg 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

<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

Claims (9)

1. A DNA molecule, characterized in that the nucleotide sequence of the DNA molecule is shown as SEQ ID NO. 1.

2. A biomaterial, comprising 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 infection;

(B) preparing the medicine for preventing the related diseases caused by SARS-CoV-2.

5. The use of claim 4, wherein said SARS-CoV-2 comprises a mutant B.1.351, a wild-type, a mutant P.1 or a mutant B.1.617 strain.

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

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

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

9. The method for producing a DNA vaccine according to any one of claims 6 to 8, 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.

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