WO2022144040A1 - Nucleotide sequence encoding novel coronavirus antigen, and use thereof - Google Patents

Abstract

Provided are a nucleotide encoding a novel coronavirus antigen, and a use thereof. The nucleotide is obtained by optimizing a wild-type DNA sequence encoding a SARS-CoV-2 virus surface spike protein, and a wild-type gene signal peptide, after being optimized, is connected to the nucleotide upstream and inserted into an eukaryotic expression vector, and then introduced into a host cell, to efficiently express a virus spike antigen in the host cell. After antigen extraction, an antiviral humoral immune response and a cellular immune response are systemically activated.

Description

A kind of nucleotide sequence encoding novel coronavirus antigen and its application

CROSS-REFERENCE TO RELATED APPLICATIONS

This disclosure claims the priority of the Chinese patent application with the application number "CN202011597679.9" and the title "A Nucleotide Sequence Encoding Novel Coronavirus Antigen and Application thereof" filed with the China Patent Office on December 29, 2020, The entire contents of which are incorporated by reference in this disclosure.

technical field

The present disclosure relates to the technical field of vaccines, in particular to a nucleotide sequence encoding a novel coronavirus antigen and applications thereof, in particular to the application in the preparation of vaccines.

Background technique

Coronaviruses are an enveloped non-segmented positive RNA virus belonging to the family Coronaviridae and the order Nidovirales, and are the largest known positive-strand RNA viruses. According to the serological and genomic characteristics of the virus, the subfamilies of coronaviruses can be divided into four genera, α, β, γ, and δ. The pneumonia of unknown cause that occurred in Wuhan in December 2019 was finally determined to be caused by a new type of coronavirus (Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2). It is spread through respiratory droplets and can also cause pneumonia (Novel Coronavirus-infected Pneumonia, NCP) through contact transmission, and the population is generally susceptible.

Since December 2019, large-scale outbreaks of SARS-CoV-2 have occurred in China and around the world, and the epidemic has caused incalculable social pressure and economic burden. On January 30, 2020, the World Health Organization declared the outbreak a "Public Health Emergency of International Concern" (PHEIC), and this public health hazard continues to spread. Although the spread of the virus in China has been effectively controlled, other countries in the world are still in a period of high incidence and even a second pandemic. According to epidemiological statistics, 20% of patients diagnosed with new crown infection require hospitalization, of which the demand ratio of ICU is 1:16,000, the case fatality rate of patients under 65 years old is 0.6-2.8%, and the case fatality rate of patients over 70 years old is 0.6-2.8%. 5.4-16.6%. As of November 2020, the World Health Organization announced that there were about 63 million confirmed cases of new coronary pneumonia worldwide and 1.46 million deaths.

These data show that the new coronavirus is still a serious threat to human health, and there is currently no effective prevention and treatment for such diseases. The development of effective preventive vaccines is an effective means to alleviate the epidemic in addition to physical isolation. Vaccines using the SARS-CoV-2 virus surface protein Spike (spike protein) as the antigen, including nucleic acid vaccines, subunit vaccines and viral vector vaccines, the expression level and protein structure of the S protein determine the effectiveness of the vaccine .

At present, there are some related researches on new coronavirus vaccines. For example, CN111218459B discloses a new coronavirus vaccine with human replication-deficient adenovirus type 5 as a carrier, which can induce the body to produce cellular and humoral immune responses in a short time, and has good immune protection effect. The new crown vaccine in this patent is a viral vector vaccine.

Nucleic acid vaccines are called "third-generation vaccines" and have the following advantages: 1. All-round induction of humoral and cellular immune responses, which can play a good preventive role; 2. Simple production process, good storage stability, and no need for cold chain Transport, suitable for large-scale application and distribution. At present, relevant nucleic acid vaccines exist. For example, patent CN110951756B discloses a nucleic acid sequence expressing SARS-CoV-2 virus antigen peptide, which can be effectively expressed in human cells and induce corresponding immune protection responses, which can be developed into SARS-CoV-2 2 The vaccine, which ensures the uniqueness of protein expression by removing potential alternative splicing sites, reduces the difficulty of subsequent purification of the protein, and further optimizes its codons by reducing the GC content to obtain the optimized nucleic acid sequence. Vaccines with high expression levels.

Since there are few types of SARS-CoV-2 nucleic acid vaccines, if we can adopt different optimization strategies from the patent CN110951756B, such as increasing the GC content in the nucleotide sequence, increasing the codon adaptation index, and increasing degenerate codon tails To obtain a nucleic acid vaccine with high expression and strong immunity for the prevention or treatment of new coronaviruses, it will greatly promote the development of the field of new coronavirus pneumonia treatment.

SUMMARY OF THE INVENTION

In view of the above deficiencies, the present disclosure provides a nucleotide sequence for encoding the S protein of a novel coronavirus (SARS-CoV-2), and the nucleotide sequence can be used to prepare a corresponding nucleic acid vaccine. Through codon-optimized design, the nucleic acid vaccine described in the present disclosure can increase the expression of its antigenic protein in vivo, and stimulate a more efficient immune response, thereby improving the preventive and/or therapeutic effect on SARS-CoV-2 virus.

The present disclosure provides a nucleotide sequence encoding a novel coronavirus antigen.

In some embodiments, the nucleotide sequence includes the coding sequence for the SARS-CoV-2 virus S protein (SARS-CoV-2 virus surface protein Spike).

In a typical embodiment, the coding sequence of the S protein of the SARS-CoV-2 virus has homology with the nucleotide sequence of the wild-type S protein, and the nucleotide sequence of the wild-type S protein is as shown in SEQ ID NO:1 shown.

In a typical embodiment, the coding sequence of the S protein of the SARS-CoV-2 virus has 65-80% homology with the nucleotide sequence of the wild-type S protein.

In a typical embodiment, the coding sequence of the S protein of the SARS-CoV-2 virus has 70-75% homology with the nucleotide sequence of the wild-type S protein.

In a typical embodiment, the third base of the codon in the coding sequence of the S protein of the SARS-CoV-2 virus is replaced by A with C or G, and T with C or G.

In a typical embodiment, the GC content of the coding sequence of the S protein of the SARS-CoV-2 virus is 40-80%.

In a typical embodiment, the GC content of the coding sequence of the S protein of the SARS-CoV-2 virus is 45-70%, optionally 50-60%.

In a typical embodiment, the coding sequence of the S protein of the SARS-CoV-2 virus is shown in SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

In some embodiments, the nucleotide sequence further includes a signal peptide sequence as shown in SEQ ID NO: 2.

In a typical embodiment, after the signal peptide sequence is optimized, the expression of the post-translation protein can be further increased by enhancing the transport of the post-translation protein between organelles, thereby enhancing the immunogenicity of the nucleic acid vaccine. The signal peptide sequence of the unoptimized S protein wild-type is shown in SEQ ID NO:10.

In some embodiments, the nucleotide sequence is shown in SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

In some embodiments, the amino acid sequence of the S protein of the SARS-CoV-2 virus is shown in SEQ ID NO:9.

The present disclosure also provides a vector comprising the above-mentioned nucleotide sequence.

In some embodiments, the vector includes, but is not limited to, plasmid, virus, phage, RNA, and optionally plasmid DNA.

In typical embodiments, the vector is capable of expressing the SARS-CoV-2 virus S protein, which is capable of eliciting an immune response in mammals.

The present disclosure also provides the application of the above-mentioned nucleotide sequence or vector in the preparation of SARS-CoV-2 nucleic acid vaccine.

The present disclosure also provides a SARS-CoV-2 nucleic acid vaccine comprising the above-mentioned nucleotide sequence or vector.

The present disclosure also provides applications of the above-mentioned nucleotide sequences, vectors or SARS-CoV-2 nucleic acid vaccines in the preparation of medicines for preventing and/or treating SARS-CoV-2 virus-related diseases.

Compared with the prior art, the positive and beneficial effects of the present disclosure are:

The present disclosure improves the GC content in the nucleotide sequence by optimizing the nucleotide sequence encoding the SARS-CoV-2 surface protein Spike, and further increases the GC content at the 5' end of the nucleotide; Codon frequency, increase the CAI index (codon adaptation index); increase the proportion of degenerate codon tails G or C, reduce the free energy of forming RNA secondary structure, reduce the proportion of Negative CIS elements, and reduce the proportion of repetitive sequences in the sequence; and By optimizing the signal peptide of the wild-type gene, the expression amount can be further increased, the optimized nucleotide sequence can be obtained, and the expression amount of the post-translation protein amount can be increased, thereby enhancing the immunogenicity of the nucleic acid vaccine. Experiments show that the optimized nucleotide sequence is made into a nucleic acid vaccine, which greatly improves the gene transcription and expression of antigenic proteins compared with the wild-type sequence, and can induce more efficient humoral and cellular immune responses after immunizing experimental animals.

In some embodiments, the optimized nucleotide sequence is inserted into a eukaryotic expression vector, and then introduced into a host cell, so that the viral Spike antigen can be highly expressed in the host cell and on the surface. Viral humoral and cellular immune responses. Antibodies produced by an activated humoral immune response can prevent virus entry, an activated cellular immune response can further clear virus-infected cells, and an activated cellular immune response can reduce potential side effects caused by "antibody-dependent enhancement" (ADE) adverse reactions.

Description of drawings

Figure 1 is the result of enzyme digestion verification of the nucleic acid vaccine candidate engineering bacteria plasmid in Example 2 of the disclosure, wherein A is the BamHI/EcoRV digestion result of the wild-type DNA plasmid pVAX1-S (WT), and B is the DNA plasmid pVAX1- The BamHI/XhoI digestion result of ADV400, C is the BamHI/XhoI digestion result of the DNA plasmid pVAX1-ADV401, and D is the BamHI/XhoI digestion result of the DNA plasmid pVAX1-ADV402;

The bands are as follows: 1. pVAX1-S (WT), 2. pVAX1-S (WT) after digestion, 3. pVAX1-ADV400, 4. pVAX1-ADV400 after digestion, 5. pVAX1-ADV401, 6. The digested pVAX1-ADV401, 7. pVAX1-ADV402, and 8. the digested pVAX1-ADV402.

Figure 2 is a graph showing the qPCR transcription detection of the disclosed candidate nucleic acid vaccine in mammalian cells, wherein A is the detection result of pVAX1-ADV400, B is the detection result of pVAX1-ADV401, and C is the detection result of pVAX1-ADV402.

FIG. 3 is a diagram showing the expression detection of mammalian cell antigen protein of candidate nucleic acid vaccines of the present disclosure, wherein A is a flowchart of flow detection, B is a flow detection result chart, and C is a flow detection result statistical chart.

Figure 4 is a diagram showing the detection of the humoral immune response of the disclosed candidate nucleic acid vaccine, wherein A is the detection result of pVAX1-ADV400, B is the detection result of pVAX1-ADV401, and C is the detection result of pVAX1-ADV402.

Figure 5 is an ELISPOT detection chart of the disclosed candidate nucleic acid vaccine cellular immune response, wherein A is the detection result of pVAX1-ADV400, B is the detection result of pVAX1-ADV401, and C is the detection result of pVAX1-ADV402.

6 is a flow cytometry diagram of the disclosed candidate nucleic acid vaccine cellular immune response, wherein A is a flow cytometry flow chart, B is a flow cytometry result graph, and C is a flow cytometry result statistical graph.

Detailed ways

The present disclosure will be described in further detail below with reference to specific embodiments. The following embodiments are not used to limit the present disclosure, but are only used to illustrate the present disclosure. The experimental methods used in the following examples, unless otherwise specified, the experimental methods that do not specify specific conditions in the examples are usually in accordance with conventional conditions, and the materials, reagents, etc. used in the following examples, unless otherwise specified, are all Commercially available.

Unless otherwise specified, "SARS-CoV-2" in this article refers to "new coronavirus".

Unless otherwise specified, "S protein" herein refers to "SARS-CoV-2 virus surface protein Spike (spike protein)".

Unless otherwise specified, "SFU" herein refers to "spot-forming unit".

Example 1: Optimization of S protein-encoding nucleic acid and vector construction

1. The principle of nucleic acid sequence optimization is: (1) According to the preference of the host cell for nucleic acid codons, degenerate codons are optimized, so that the optimized sequence contains more nucleic acid codons that are conducive to host cell recognition; (2) In On the basis of codon preference optimization, the GC content in the nucleic acid sequence is further optimized, so that the sequence after GC content optimization can express more target proteins; (3) The nucleic acid sequence is optimized so that it can transcribe more stable mRNA, which is beneficial to the target protein. protein translation; (4) changing the codon frequency of the host preference and increasing the CAI index (codon adaptation index).

Optimization purpose: to increase the protein expression of the target protein in host cells.

Optimization strategy: Optimize the third base of amino acid-encoding codons from A to C or G, or T to C or G; increase the ratio of G or C at the tail of degenerate codons, and reduce the free energy of forming RNA secondary structure, Reduce the proportion of Negative CIS elements and reduce the proportion of repetitive sequences in the sequence.

Optimization results: only the nucleic acid sequence was changed, but the amino acid sequence was not changed.

Optimization step: Select the SARS-CoV-2 virus surface protein Spike (S protein) as the antigen, and its amino acid sequence is shown in SEQ ID NO: 9, and the wild-type nucleic acid coding sequence SWT (SEQ ID NO. : 1) carry out optimization to obtain the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, and combine the optimized sequence with the optimized signal peptide sequence (SEQ ID NO: 5). : 2) connect to obtain 3 nucleotide sequences named ADV400 (SEQ ID NO: 6), ADV401 (SEQ ID NO: 7) or ADV402 (SEQ ID NO: 8) respectively, and then carry out the optimized sequence synthesis.

By optimizing the obtained sequences, in terms of GC content, ADV400, ADV401, and ADV402 sequences increased the GC content at the 5' end of DNA (60%), while the GC content at the same site of the wild-type sequence was less than 50%.

2. The 3 nucleotide sequences obtained above, and the wild-type coding sequence of the S protein were transformed and constructed into the pVAX1 carrier (ThermoFisher, article number: V26020), respectively, to obtain plasmid DNAs: pVAX1-S (WT), pVAX1- ADV400, pVAX1-ADV401 and pVAX1-ADV402.

Example 2: Construction of nucleic acid vaccine (each optimized sequence)

1. Transformation of nucleic acid vaccine candidate sequences

(1) Take 100 μL of the competent cell (such as DH5α) suspension from the -70°C refrigerator and thaw on ice.

(2) The plasmid DNA solutions prepared in Example 1 (volume not exceeding 10 μL): pVAX1-S (WT), pVAX1-ADV400, pVAX1-ADV401 and pVAX1-ADV402 were added to the competent cells, and shaken gently. , placed on ice for 30min.

(3) Heat shock in a water bath at 42°C for 90s, then quickly cool on ice for 5min.

(4) 1 mL of LB liquid medium (without antibiotics) was added to the tube, and after mixing, it was shaken and cultured at 37° C. for 45 minutes to restore the normal growth state of the bacteria.

(5) Shake 100 μL of the above bacterial liquid and spread it on a screening plate containing appropriate antibiotics, place it face up, turn the petri dish upside down after the bacterial liquid is completely absorbed by the medium, and cultivate at 37°C for 8-16 hours.

(6) Pick a single colony of cells with an even edge, and use a pipette tip to pick the clone and place it in 5 mL of LB selection medium for overnight culture at 37°C.

2. Plasmid extraction of nucleic acid vaccine candidate engineering bacteria:

(1) Centrifuge at 12000 rpm for 5 min to collect bacterial cells.

(2) 1 mL of solution I (25 mM Tris-HCl, pH 8.0, Sigma, cat. No: T1819; 10 mM EDTA, Sigma, cat. No: T3913) was added to make the cells completely and uniformly dissolved in the solution.

(3) Add solution II (0.4N NaOH, Beijing Chemical Reagents Company, cat. No: 10019792; 2% SDS, Sigma, cat. No: L5750) according to the ratio of solution II: solution I = 1:1, spin and centrifuge carefully tube to mix the solution thoroughly.

(4) Add solution III (3M KAc, 5M HAc, Beijing Chemical Reagents Company, cat. No: 30154592, cat. No: 10000292) with a volume of 1.5 times the volume of solution I, gently shake and mix.

(5) Centrifuge at 12000 rpm for 10 min, take the supernatant and add 60% volume of isopropanol, invert up and down to mix, and let stand at room temperature for 15 min.

(6) Centrifuge at 12000 rpm for 10 min, discard the supernatant, and finish the rough extraction.

(7) Wash with 70% ethanol, blow dry, and dissolve the precipitate with an appropriate amount of TE.

3. Identification of nucleic acid vaccine candidate engineered bacteria plasmids by enzyme digestion:

The candidate plasmids pVAX1-ADV400, pVAX1-ADV401 and pVAX1-ADV402 extracted in step 2 were digested with BamHI/XhoI double enzymes for specific restriction fragment identification, and pVAX1-S (WT) was specifically digested with BamHI/EcoRV double enzymes The restriction enzyme digestion fragment was identified; the restriction enzyme digestion system is as shown in Table 1, and the digestion reaction was carried out at 37°C for 4 hours. After digestion, electrophoresis was performed on a 1% agarose gel.

Table 1 Double enzyme digestion reaction system

reagent	Volume (μL)
pVAX1-ADV	10
BamHI	0.5
XhoI or EcoRV	0.5
10×buffer	2
Deionized water	7
total capacity	20

The results are shown in FIG. 1 , the plasmids pVAX1-ADV400, pVAX1-ADV401 and pVAX1-ADV402 constructed by the nucleic acid vaccine described in the present disclosure are all correct.

Example 3: Transcriptional identification of candidate nucleic acid vaccine mammalian cells

1. In vitro transfection of nucleic acid vaccines

(1) Take out the cryopreserved HEK293 cell line (ATCC, CRL-3216) from liquid nitrogen, and then centrifuge at 1000 rpm for 5 min at 37 °C in a water bath to remove DMSO;

(2) Add serum-free DMEM medium for washing once, and incubate in 5 mL of DMEM medium containing 10% calf serum at 37°C, 5% CO ₂ . 24h before transfection, cells were digested with trypsin (0.25%) at 37°C for 5min and terminated, then plated at a density of (1-3)×105 cells/well in a ⁶ -well plate or 35mm culture dish, and 2mL of growth culture was added. culture in a 37°C, 5% CO ₂ incubator for 20-24h, until the cell density is 50-80%;

(3) pVAX1-ADV400, pVAX1-ADV401 and pVAX1-ADV40, as well as the wild-type pVAX1-S (WT), the empty plasmid pVAX1, were transformed at the same time for parallel experiments. Add 100 μL of serum-free DMEM medium to each, mix gently, and place at room temperature for 10 min;

(4) Put 2 μL of cationic liposomes in 100 μL of serum-free DMEM medium, mix gently, and place at room temperature for 10 minutes;

(5) Mix the above plasmid and liposome, mix gently, and place at room temperature for 15-30min.

(6) Aspirate the medium in the culture plate/dish, wash it once with 2 mL of serum-free medium, add 0.8 mL of serum-free medium to each well or 35mm culture dish, and then add the above DNA plasmid/lipid dropwise body complexes and distribute them evenly. Incubate at 37°C, 5% _CO2 incubator for 4-8h, typically 6h.

(7) After culturing for 6 h, remove the mixed liquid, add complete medium and continue to incubate for 24 h.

2. Post-transfection RNA extraction

(1) Collect the cells after transfection and digestion, centrifuge at 4000 rpm for 5 min, discard the supernatant and resuspend the cells in 50 μL of PBS;

(2) Add 1 mL of TRIzol, invert and mix for 10 times, and incubate at room temperature for 5 min;

(3) Add 1/5 volume of chloroform (0.2mL), invert and mix for 15s, and incubate at room temperature for 5min;

(4) Centrifuge at 12000rpm at 4°C for 15min, transfer the upper aqueous phase (about 400μL) to another 1.5mL EP tube.

(5) Add an equal volume of isopropanol, mix at room temperature for 10 minutes, and centrifuge at 12000 rpm for 10 minutes at 4°C.

(6) Discard the supernatant, add 1 mL of ice-cold 75% ethanol (prepared with DEPC water), and centrifuge at 12000 rpm at 4°C for 5 min.

(7) Discard the supernatant, air dry for 5-10 min, and add 20 μL of RNase-free sterile water to dissolve the RNA.

3. RNA reverse transcription, qPCR reaction

(1) Prepare a solution according to the number of PCRs required to be n (n=number of samples+1 tube of negative control+1 tube of positive control).

(2) Prepare the reaction system in Table 2 below.

Table 2 Reaction system

reagent	Volume (μL)
RT Buffer 5×	4
dNTP mix(10μM each)	0.5
Oligo dT	1

Total RNA	5
RTase	1
RNase-free sterile water	8.5

(3) Perform reverse transcription according to the reaction program in Table 3 below.

Table 3 Reaction program

temperature	time
30℃	10min
42℃	1h
99℃		5min
4℃	store

(4) After the reverse transcription, the reaction system was prepared according to Table 4.

Table 4 Reaction system

(5) Carry out qPCR reaction according to the amplification procedure in Table 5 below.

Table 5 Reaction program

The results are shown in FIG. 2 , and it can be seen from FIG. 2 that the nucleic acid vaccines prepared by the optimized nucleotide sequences ADV400, ADV401 and ADV402 in the present disclosure, namely pVAX1-ADV400, pVAX1-ADV401 and pVAX1-ADV402, compared with wild The nucleic acid vaccine pVAX1-S (WT) prepared with the nucleotide sequence of the same type, and the empty plasmid pVAX1, have significantly increased transcriptional effects in mammalian cells.

Example 4: Identification of candidate nucleic acid vaccine mammalian cell antigen protein expression

1. Carry out transfection according to the same operation as step 1 in Example 3. After 24 hours of transfection, remove the culture medium after transfection, wash with pre-cooled PBS, and discard PBS.

2. Preparation of single cell suspension. After counting, cells were plated in a 96-well round bottom plate according to the number of 1×10 ⁶ cells/well, to be used for staining.

3. Centrifuge the 96-well plate containing the single cell suspension at 1500 rpm/min for 3 min, and discard the supernatant.

4. Add the prepared cell death dye to the staining buffer according to the corresponding amount, the staining buffer is 2% FBS/PBS, and the staining volume is 50 μL per well. Gently blow and suck 3-5 times. Dyeing at room temperature in the dark for 15 min.

5. Centrifuge the 96-well plate containing the single cell suspension at 1500 rpm/min for 3 min, and discard the supernatant.

6. Add the anti-Spike flow antibody directed against the cell surface to the staining buffer in a staining volume of 50 μL per well. Resuspend the cells in the 96-well plate with the surface antibody, and gently pipette 3-5 times. Dyeing at room temperature and dark for 45min.

7. Add 200 μL of staining buffer to each well to terminate the staining, protect from light, and centrifuge the 96-well plate where the single cell suspension is located at 1500 rpm/min for 3 min, and discard the supernatant.

8. Add 200 μL of staining buffer to each well to resuspend, and gently pipette 3-5 times. Centrifuge the 96-well plate where the single cell suspension is located at 1500 rpm/min for 3 min, and discard the supernatant.

9. Add 200 μL of staining buffer to each well to resuspend, gently pipette 3-5 times, and wait for the flow cytometer to detect the loading.

10. When large particles are turbid, they should be filtered through a 200-mesh copper mesh again before loading on the machine.

The detection results are shown in FIG. 3 . It can be seen from FIG. 3 that the nucleic acid vaccines prepared from the optimized nucleotide sequences ADV400, ADV401 and ADV402 in the present disclosure, namely pVAX1-ADV400, pVAX1-ADV401 and pVAX1-ADV402 , compared with the nucleic acid vaccine pVAX1-S (WT) prepared by wild-type nucleotide sequence, the expression of antigenic protein in mammalian cells was significantly increased.

Example 5: Validation of candidate nucleic acid vaccine immunogenicity

In order to verify the immunogenicity of the nucleic acid vaccine prepared in Example 2, the prepared nucleic acid vaccine was used to immunize mice twice every two weeks. The first day of vaccine immunization was counted as day 0. At the same time, buffer solution was used as a control, which was recorded as SSC.

In order to further evaluate the antigen-specific cellular responses elicited by nucleic acid vaccines, a S antigen-specific peptide pool MHC-I/MHC-II epitope peptide (S peptide) was predicted and synthesized, and the peptide pool was used to stimulate small cells from post-vaccine immunization. Splenocytes or lymph node cells isolated from mice. The mice used in this experimental example were 6-8 week old BALB/c mice, which were purchased from Beijing Huafukang Company. Some experimental methods or conditions are listed as follows:

Enzyme-linked immunosorbent assay for humoral immune response: blood samples were collected from mice on the 14th day, and the specific antibody titers of the serum were determined by ELISA. When it is necessary to quantitatively detect the antibody concentration in mouse serum, a standard curve is added on the basis of conventional ELISA, and the concentration of antibody in mouse serum is determined according to the concentration of the standard curve.

The detection results are shown in Figure 4, and it can be seen from Figure 4 that the optimized nucleotide sequences ADV400, ADV401 and ADV402 in the present disclosure, the prepared nucleic acid vaccines, namely pVAX1-ADV400, pVAX1-ADV401 and pVAX1-ADV402, Compared with the nucleic acid vaccine pVAX1-S(WT) prepared with wild-type nucleotide sequence, the concentration of antibody in the serum of mice was significantly increased.

Detection of cellular immune responses by immune cell-specific stimulation: performed in a sterile environment, the mice were de-necked, euthanized, the spleen or lymph nodes were removed, and ground into a single-cell suspension; cells were harvested by centrifugation, and the red blood cell lysate was resuspended and then lysed with FBS. PBS to terminate the lysis; filter, count the prepared single cell suspension, and plate with 1×10 ⁶ cells/well; add the corresponding specific polypeptide pools according to ELISPOT and flow cytometry respectively for in vitro stimulation, ELISPOT at 37°C, Detection was performed after 24h incubation in 5% CO ₂ . Flow assay was cultured at 37°C, 5% CO ₂ for 6 h, and the stimulated cells were collected by centrifugation. Flow cytometry detection.

The ELISPOT detection results are shown in Figure 5, and the flow detection results are shown in Figure 6, indicating that the optimized nucleotide sequences ADV400, ADV401 and ADV402 in the present disclosure, the prepared nucleic acid vaccines, namely pVAX1-ADV400, pVAX1- Compared with the nucleic acid vaccine pVAX1-S(WT) prepared with wild-type nucleotide sequence, the specificity of Spike antigen was significantly increased for ADV401 and pVAX1-ADV402.

In summary, the present disclosure improves the GC content in the nucleotide sequence by optimizing the nucleotide sequence encoding the SARS-CoV-2 surface protein Spike, and further increases the GC content at the 5' end of the nucleotide; at the same time, changing the host Preferential codon frequency increases the CAI index; increases the proportion of degenerate codon tails G or C, reduces the free energy of forming RNA secondary structures, reduces the proportion of Negative CIS elements, and reduces the proportion of repetitive sequences in the sequence. The optimized gene signal peptide can be further improved, and the optimized nucleotide sequence can be obtained and made into a nucleic acid vaccine. Experiments have shown that the nucleic acid vaccine prepared by the optimized nucleotide sequence greatly improves the gene transcription and expression of the antigenic protein compared with the wild-type sequence, and can induce more efficient humoral and cellular immune responses after immunizing experimental animals. .

The above-mentioned embodiments only represent several embodiments of the present disclosure, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the scope of the present disclosure. It should be noted that, for those skilled in the art, without departing from the concept of the present disclosure, several modifications and improvements can be made, which all belong to the protection scope of the present disclosure. Accordingly, the scope of protection of the present disclosure should be determined by the appended claims.

Claims (10)

1. A nucleotide sequence encoding a novel coronavirus antigen, characterized in that the nucleotide sequence comprises the coding sequence of the S protein of the SARS-CoV-2 virus;

   The coding sequence of the S protein of the SARS-CoV-2 virus has homology with the nucleotide sequence of the wild-type S protein, and the nucleotide sequence of the wild-type S protein is shown in SEQ ID NO: 1.
2. The nucleotide sequence according to claim 1, wherein the coding sequence of the S protein of the SARS-CoV-2 virus has 65-80% homology with the nucleotide sequence of the wild-type S protein, It is preferably 70-75%.
3. The nucleotide sequence according to claim 1 or 2, wherein the third base of the codon in the coding sequence of the S protein of the SARS-CoV-2 virus is replaced by C or G, T is C or G;

   The GC content of the coding sequence of the S protein of the SARS-CoV-2 virus is 40-80%, preferably 45-70%, more preferably 50-60%.
4. The nucleotide sequence according to claim 3, wherein the coding sequence of the S protein of the SARS-CoV-2 virus is as shown in SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 .
5. The nucleotide sequence according to claim 4, wherein the nucleotide sequence further comprises a signal peptide sequence as shown in SEQID NO:2.
6. The nucleotide sequence according to claim 5, wherein the nucleotide sequence is shown in SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.
7. A vector, characterized in that the vector comprises the nucleotide sequence of any one of claims 1-6.
8. Application of the nucleotide sequence according to any one of claims 1-6 or the vector according to claim 7 in the preparation of a SARS-CoV-2 nucleic acid vaccine.
9. A SARS-CoV-2 nucleic acid vaccine, characterized in that the nucleic acid vaccine comprises the nucleotide sequence described in any one of claims 1-6 or the vector described in claim 7.
10. The nucleotide sequence according to any one of claims 1-6, the vector according to claim 7 or the nucleic acid vaccine according to claim 9 in the preparation of a medicine for preventing and/or treating SARS-CoV-2 virus-related diseases Applications.

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