Novel trivalent DNA vaccine for coronavirus and preparation method thereof

Abstract

The present application relates to the technical field of novel coronavirus, and particularly relates to a novel coronavirus trivalent DNA vaccine and a preparation method. The trivalent DNA vaccine takes a recombinant plasmid carrying a nucleotide sequence as shown in SEQ ID NO: 1 or 2 as an active ingredient. The trivalent DNA vaccine provided in the present application can express a fusion antigen protein of three kinds of antigen genes of the novel coronavirus in eukaryotic cells, and produce serum IgG antibodies against the three kinds of antigens of the novel coronavirus. In addition, the novel coronavirus trivalent DNA vaccine provided in the present application can immunize mice to produce T cells secreting various cytokines, and has a stronger T cell immune protection effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of novel coronavirus, in particular to a novel coronavirus trivalent DNA vaccine and a preparation method thereof. BACKGROUND

[0002] The novel coronavirus is a single-strand RNA virus with a capsid, which encodes 16 non-structural proteins, 9 auxiliary proteins and 4 major structural proteins. Among them, the structural proteins are envelope protein, membrane protein, nucleocapsid protein and spike protein. The S protein in the form of homotrimer exists in the viral envelope and is composed of S1 and S2 functional subunits. The S1 subunit contains a receptor binding region, which is responsible for recognizing the receptor-angiotensin converting enzyme 2 on the surface of human host cells. The S2 subunit contains a fusion peptide, a linker region, a heptad repeat, a central helix, etc., which mediates the fusion of the virus and the host cell membrane. When the S1 subunit binds to the host cell surface receptor ACE2, the host protease recognizes and cleaves the S1 / S2 cleavage site, the S1 subunit falls off, and the FP protruding after the S2 subunit changes in conformation performs membrane fusion. Studies have shown that the RBD region of the S protein of the novel coronavirus has immunogenicity and is the target of 90% of neutralizing antibodies in immune serum.

[0003] To obtain more comprehensive protection against the novel coronavirus, multiple antigen proteins can often be fused and expressed as a fusion protein vaccine, a fusion DNA vaccine, etc. to obtain the effect of multiple protection with one immunization. However, fusion expression does not necessarily mean that multiple antibodies or cross-protection immune effects can be induced, and fusion protein expression can interfere with each other, and the immune effects of multiple antigens can interfere with each other and reduce or inhibit the immune effects of one or several antigens, thereby failing to achieve the effect of multiple protection with one immunization. SUMMARY

[0004] To solve or partially solve the problems in the related art, the present application provides a novel coronavirus trivalent DNA vaccine and a preparation method thereof.

[0005] The first aspect of the present application provides a nucleic acid as shown in SEQ ID NO: 1 or 2.

[0006] The second aspect of the present application provides a recombinant plasmid carrying a nucleotide sequence as shown in SEQ ID NO: 1 or 2.

[0007] In some embodiments, the recombinant plasmid is a pCI-neo plasmid carrying a nucleotide sequence as shown in SEQ ID NO: 1 or 2.

[0008] The third aspect of the present application provides a method for preparing the recombinant plasmid of the second aspect. The method comprises connecting the target fragment as shown in SEQ ID NO: 1 or 2 with linearized pCI-neo, transferring the connected fragment into E. coli, screening positive clones from the transformants, and extracting the pCI-neo plasmid carrying the target fragment as shown in SEQ ID NO: 1 or 2 from the positive clones.

[0009] In some embodiments, the step of connecting the target fragment as shown in SEQ ID NO: 1 or 2 with linearized pCI-neo comprises: PCR amplifying the target fragment as shown in SEQ ID NO: 1 or 2 carrying the end of NheI enzyme cutting site and SalI enzyme cutting site; using NhoI and SalI to respectively cut the pCI-neo plasmid to obtain linearized pCI-neo; and connecting the target fragment as shown in SEQ ID NO: 1 or 2 carrying the end of NheI enzyme cutting site and SalI enzyme cutting site with linearized pCI-neo respectively.

[0010] In some embodiments, the preparation process of the linearized pCI-neo fragment comprises: taking 200 ng of pCI-neo plasmid, 2 μL of 10 U / μL NhoI, 10 μL of M-buffer, and 38 μL of deionized water, mixing uniformly, reacting in a 37℃ water bath for 120 min, and purifying; taking 45 μL of the purified NhoI enzyme cut pCI-neo fragment, 10 μL of 10× buffer, 8 μL of 1.7 mM dNTP, 1 μL of Taq DNA polymerase, and 36 μL of deionized water, reacting in a 37℃ water bath for 60 min, purifying, and obtaining an intermediate product; taking 48 μL of the intermediate product, 10 μL of 10× Hbuffer, 2 μL of Sal I, and 40 μL of deionized water, reacting in a 37℃ water bath for 120 min, purifying, electrophoresis, and gel recovery, and obtaining the linearized pCI-neo fragment.

[0011] In some embodiments, the step of connecting reaction comprises: linearizing the pCI-neo fragment carrying the NheI enzyme cutting site and the SalI enzyme cutting site at the end of the target fragment as shown in SEQ ID NO: 1 or 2, respectively, mixing the linearized fragments at a molar ratio of 5:1, performing directional ligation for 30 min at 12 μL of reaction system using ligation I connecting enzyme at 16°C, transforming the ligation product into competent E. coli Top10, inoculating the transformed competent cells on the surface of LB agar plates containing ampicillin, and culturing at 37°C overnight to obtain colonies; randomly picking single colonies with toothpicks and transferring them into 12 LB culture mediums containing 2 mL of ampicillin, and culturing at 37°C overnight; adding 100 μL of the screened positive bacterial liquid into a flask containing 50 mL of LB culture medium with ampicillin and culturing at 30°C, large-scale extraction and purification, measuring the purity and concentration with a UV instrument, and storing at -20°C for standby; extracting the plasmid from the bacterial liquid, and screening the recombinant plasmid by double enzyme digestion with restriction endonuclease NheI and SalI.

[0012] The fourth aspect of the present application provides a novel coronavirus trivalent DNA vaccine, which takes the pCI-neo plasmid as shown in SEQ ID NO: 1 or 2 as an active ingredient.

[0013] In some embodiments, the novel coronavirus trivalent DNA vaccine further comprises 1-3.4% of a polyvinylpyrrolidone aqueous solution.

[0014] The fifth aspect of the present application provides the nucleic acid of the first aspect and the recombinant plasmid of the second aspect for preparing a novel coronavirus vaccine.

[0015] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present application.

[0016] The present application has the following beneficial technical effects:

[0017] The present application selects special purpose sequences from the novel coronavirus genome to synthesize purpose sequence 1, purpose sequence 2 and purpose sequence 3, and clones these purpose sequences into the eukaryotic expression plasmid pCI-neo, and then performs eukaryotic cell expression of the fusion antigen protein of the three antigen genes in Vero cells. It is found that the eukaryotic expression plasmid pCI-neo carrying the purpose sequence 1 and the purpose sequence 2 can express a fusion protein in Vero cells that can be detected by Anti-Spike antibody, Anti-N antibody and nsp12 / RdRP antibody, indicating that it has immunogenicity against multiple antigens of novel coronavirus spike protein, N protein and RdRP protein. The eukaryotic expression plasmid pCI-neo carrying the purpose sequence 3 expresses a fusion protein in Vero cells that can only be detected by nsp12 / RdRP antibody. Therefore, the antigen protein expression of the monoclonal cells carrying pCI-TV-RNR1 and pCI-TV-RNR2 is normal, while the antigen protein expression of the monoclonal cells carrying pCI-TV-RNR3 may only have affinity for nsp12 / RdRP antibody or its affinity for Anti-Spike antibody or Anti-N antibody is reduced, or it does not have immunogenicity against novel coronavirus spike protein and N protein.

[0018] In addition, the present application also immunizes mice with eukaryotic expression plasmid pCI-neo carrying purpose sequence 1, purpose sequence 2 and purpose sequence 3, and the results show that the recombinant plasmid carrying purpose sequence 1 and purpose sequence 2 can produce serum IgG antibodies against S1 spike protein, N protein or RdRP protein of novel coronavirus, and has stronger protective effect. The recombinant plasmid carrying purpose sequence 3 only detects RdRP protein serum IgG antibodies in the serum of the 28th day, and cannot detect serum N protein serum IgG antibodies and a small amount of S1 spike protein serum IgG antibodies, and its protective effect is obviously inferior to recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2. Therefore, it is suggested that recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2 can both develop into novel coronavirus vaccines, and the protective effect of recombinant plasmid pCI-TV-RNR2 is stronger.

[0019] In addition, the immunized mice containing the recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2 can produce T cells secreting various cytokines, and the T cells secreting cytokines of the immunized product of the recombinant plasmid pCI-TV-RNR2 have a higher concentration. The T cell immunity can recognize and eliminate pathogens, and help the body to recover health. Therefore, it is suggested that the recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2 can both develop into a new coronavirus vaccine, and the recombinant plasmid pCI-TV-RNR2 has a stronger protective effect, and has a stronger T cell immune protection effect.

[0020] In addition, the test shows that the recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2 provided by the application can produce sufficient immune protection effect of killing and eliminating the new coronavirus in the lungs of mice, and the recombinant plasmid pCI-TV-RNR2 immunized mice have the strongest elimination effect. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The WB detection results of the serum IgG antibodies against the S1 spike protein, N protein or RdRP protein of the new coronavirus of the fusion proteins expressed in Vero cells by the pCI-neo, pCI-TV-RNR1, pCI-TV-RNR2 and pCI-TV-RNR3 plasmids, respectively.

[0022] Figure 2 The flowchart of immunizing mice with the sample liquid containing the pCI-neo, pCI-TV-RNR1, pCI-TV-RNR2 and pCI-TV-RNR3 plasmids, respectively.

[0023] Figure 3 The detection results of the serum IgG antibody levels against the S1 spike protein, N protein or RdRP protein of the new coronavirus in the serum of the mice immunized with the sample liquid containing the pCI-neo, pCI-TV-RNR1, pCI-TV-RNR2 and pCI-TV-RNR3 plasmids, respectively.

[0024] Figure 4 The quantitative analysis results of the T cell secreted cytokines (IFN-γ, TNF-α, IL-2, IL-6, IL-4 and IL-10) in the peripheral blood mononuclear cells of the mice immunized with the sample liquid containing the pCI-neo, pCI-TV-RNR1, pCI-TV-RNR2 and pCI-TV-RNR3 plasmids, respectively.

[0025] Figure 5HE staining images of liver, lung, heart, kidney, brain and spleen tissues of mice immunized with sample liquid containing pCI-neo, pCI-TV-RNR1, pCI-TV-RNR2 and pCI-TV-RNR3 plasmids, respectively.

[0026] Figure 6 Results of detection of neutralizing antibody levels in animal level challenge experiment of serum of mice immunized with sample liquid containing pCI-neo, pCI-TV-RNR1, pCI-TV-RNR2 and pCI-TV-RNR3 plasmids on day 28, respectively.

[0027] Figure 7 Results of detection of new coronavirus genome RNA copy number in lung tissues in animal level challenge experiment of serum of mice immunized with sample liquid containing pCI-neo, pCI-TV-RNR1, pCI-TV-RNR2 and pCI-TV-RNR3 plasmids on day 28, respectively. DETAILED DESCRIPTION

[0028] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application. The reagents not described in detail in the present application are conventional reagents and can be obtained from commercial channels; the methods not described in detail are conventional experimental methods and can be known from prior art.

[0029] 1, Target fragment

[0030] In an example, the target sequence 1 (such as SEQ ID NO: 1), the target sequence 2 (such as SEQ ID NO: 2) and the target sequence 3 (such as SEQ ID NO: 3) are obtained by chemical synthesis or conventional PCR method.

[0031] 2, Recombinant plasmid

[0032] In an example, the target sequence 1 is obtained by amplifying the target sequence 1 using F1 and R1 as a primer pair, the target sequence 1 is connected with a linearized pCI-neo fragment, the connected fragment is transferred into E. coli, and a positive clone is screened from the transformant, and the pCI-neo recombinant plasmid carrying the target sequence 1 is obtained by extraction from the positive clone. Wherein, F1 is: ccgGCTAGCagagtccaaccaacagaatctattgttagatttc, the capital letters are the recognition sequence of NheI enzyme, and SEQ ID NO: 4. R1 is: cgGTCGACtttaactagcattgtatgttgagagcaaaattcatgaggtcc, the capital letters are the recognition sequence of SalI enzyme, and SEQ ID NO: 5.

[0033] One embodiment uses F2 and R2 as a primer pair to amplify the target sequence 2 to obtain the target fragment 2, connects the target fragment 2 with the linearized pCI-neo fragment, transforms the connected fragment into E. coli, screens positive clones from the transformants, and extracts the pCI-neo recombinant plasmid carrying the target sequence 2 from the positive clones. F2 is ccgGCTAGCtctaacaatcttgattctaaggttggtgg, the capital letters are the recognition sequence of NheI enzyme, and SEQ ID NO: 6. R2 is cgGTCGACtttaactagcattgtatgttgagagcaaaattcatgaggtcc, the capital letters are the recognition sequence of Sail enzyme, and SEQ ID NO: 7.

[0034] One embodiment uses F3 and R3 as a primer pair to amplify the target sequence 3 to obtain the target fragment 3, connects the target fragment 3 with the linearized pCI-neo fragment, transforms the connected fragment into E. coli, screens positive clones from the transformants, and extracts the pCI-neo recombinant plasmid carrying the target sequence 3 from the positive clones. F3 is ccgGCTAGCagagtccaaccaacagaatctattgttagatttc, the capital letters are the recognition sequence of NheI enzyme, and SEQ ID NO: 8. R3 is cgGTCGACggcccctaggattcttgatggatctgggtaaggaaggtacacataa, the capital letters are the recognition sequence of Sail enzyme, and SEQ ID NO: 9.

[0035] The reaction system of PCR amplification is 10 μL 5×transPfu buffer, 2.5 mM 5 μL dNTP, 1 μL Pfu DNA polymerase, 1.5 μL upstream primer, 1.5 μL downstream primer, 1 μL target sequence 1, 2 or 3, and 50 μL of enzyme-free double distilled water. The reaction conditions are 95°C for 5 min, 95°C for 40 s, 57°C for 40 s, 72°C for 1.5 min for 35 cycles, 72°C for 10 min, and 4°C for reaction termination.

[0036] The above target fragments 1, 2 and 3 can be obtained by other primers or chemical synthesis method, and are not limited to the steps of the above embodiments.

[0037] The preparation process of the linearized pCI-neo fragment is as follows:

[0038] Take 200 ng of pCI-neo plasmid (promega company), 2 μL of 10 U / μL NhoI (ER0971, Thermo Scientific TM), M-buffer 10 μL and deionized water 38 μL were mixed uniformly, and reacted in a 37°C water bath for 120 min, and purified according to the operating manual of Cycle-pure Kit (Omega Bio-tek). Then, 45 μL of the purified Nho I enzyme-digested pCI-neo fragment, 10 x buffer 10 μL, 1.7 mM dNTP 8 μL, 1 μL Taq DNA polymerase and deionized water 36 μL were reacted in a 37°C water bath for 60 min to obtain an intermediate product. 48 μL of the intermediate product, 10 μL of 10 x Hbuffer, Sal I 2 μL and deionized water 40 μL were reacted in a 37°C water bath for 120 min. Purification, electrophoresis and gel recovery were performed to obtain the linearized pCI-neo fragment.

[0039] The step of connecting the target fragment 1, the target fragment 2 or the target fragment 3 with the linearized pCI-neo fragment, respectively, is as follows:

[0040] The target fragment 1, the target fragment 2 or the target fragment 3 and the linearized pCI-neo fragment were mixed at a molar ratio of 5:1, and were connected with ligation I connection enzyme at 12 μL in a 16°C water bath for 30 min. The connection product was transformed into competent E. coli Top10, and the transformed competent cells were inoculated on the surface of LB agar plates containing ampicillin. After incubation at 37°C overnight, colonies appeared. A toothpick was used to randomly select single colonies and inoculate into 12 LB culture medium containing 2 mL of ampicillin, and incubated at 37°C with shaking (200 rpm) overnight. 100 μL of the selected positive bacterial solution was added to 50 mL of LB culture medium containing ampicillin in a flask and incubated at 30°C with shaking overnight. Large-scale extraction and purification were performed. The purity and concentration were measured by a UV instrument, and the product was stored at -20°C for standby. The plasmid was extracted from the bacterial solution, and the pCI-neo recombinant plasmid carrying the target sequence 1, the pCI-neo recombinant plasmid carrying the target sequence 2 or the pCI-neo recombinant plasmid carrying the target sequence 3 were screened by double digestion with restriction endonucleases Nhe I and Sal I. The rapid ligation kit (DNA ligation Kit) and the plasmid DNA extraction kit, purification kit were purchased from Sigma Company, USA. TM Kit) and the plasmid DNA extraction kit, purification kit were purchased from Sigma Company, USA.

[0041] 3. Eukaryotic cell expression of the target sequence

[0042] (1) Linearization of the recombinant plasmid

[0043] The plasmid PCI-neo was linearized by restriction enzyme BamH I for transfection: 100 μL of PCI-neo, 15 μL of buffer, 6 μL of BamH I and 29 μL of deionized water were mixed uniformly and reacted in a 37 °C water bath for 120 min. The purification was the same as above. The purity and concentration were measured on a UV instrument.

[0044] (2) Resuscitation and culture of Vero cells

[0045] The Vero cell cryopreservation tube (Cat. No. CL-0242, Wuhan Ponsay) was quickly taken out from liquid nitrogen and put into a 37 °C water bath for shaking to completely dissolve it within 1 min. The cell suspension was transferred into a centrifuge tube and centrifuged at 1000 r / min for 3 min. The supernatant was discarded, 3 mL of DMEM was added to completely dissolve the cells, and then the mixture was transferred into a 25 cm 3 glass bottle. About 10 mL of RPMI-1640 complete culture medium was added, and the culture was carried out in a 37 °C, 5% CO2 incubator. The medium was changed every 2-3 days. Vero cells are adherent cells, and after the cells covered the glass bottle, they were digested and passaged.

[0046] (3) Transfection of Vero cells

[0047] The cells were inoculated one day before transfection, and the cells should reach 60-80% coverage on the day of transfection. Generally, 3 x 10 5Cells. Fresh serum-free medium was replaced 2h before the addition of the liposome / DNA mixture on the day of transfection. Liposome / DNA mixture was prepared. Solution A: 2.5μg of linearized PCI-neo recombinant plasmid (carrying sequence 1, 2 or 3, respectively) was prepared in 20mM HEPES (pH 7.4) to a total volume of 25μL. Solution B: Lipofectamine was diluted in 20mM HEPES (pH 7.4) to a total volume of 35μL. Solutions A and B were gently mixed (pipette up and down several times, do not vortex) and incubated at room temperature for 15min to allow the liposome / DNA mixture to form. The medium was not removed and 60μL of the liposome / DNA mixture was added dropwise while gently rocking the plate. The plate was incubated at 37°C for 6h. After 6h the medium was replaced with 2mL of fresh serum-containing growth medium. Twenty-four hours after transfection, the medium was replaced with G418-containing medium (400μg / mL). The medium was replaced once 3 days later, the G418 concentration was increased to 600μg / mL 5 days later, and to 800μg / mL 8 days later. Surviving cells in the 6-well plates were transferred to culture flasks and maintained in 400μg / mL G418. The cells were selected for another 20 days or so until they grew stably and were expanded.

[0048] (4) Western blot

[0049] Western blot was used to identify the expression of sequence 1, sequence 2 and sequence 3 in the monoclonal cell lines. Monoclonal cells transfected with recombinant plasmid (pCI-TV-RNR1) carrying sequence 1, monoclonal cells transfected with recombinant plasmid (pCI-TV-RNR2) carrying sequence 2, and monoclonal cells transfected with recombinant plasmid (pCI-TV-RNR3) carrying sequence 3.

[0050] The 12th and 15th generation monoclonal cell lines were selected, the RIPA lysis buffer was used to fully lyse the monolayer of screened cells, the quantitative protein was taken for SDS-PAGE separation of different size proteins, the separated proteins were transferred to PVDF membrane by transmembrane instrument, the PVDF membrane was washed with TBST buffer (pH = 7.4, 2‰ Tween 20) for 3 times, 4% skim milk was sealed at room temperature for 2h. Incubate with primary antibody at 4℃ overnight. Wash 3 times with TBST buffer, dilute HRP-labeled goat anti-mouse secondary antibody at 1:500, incubate at room temperature for 2h. Wash 3 times with TBST buffer and observe after DAB substrate color development. The primary antibody is Anti-Spike antibody (GenScript, Cat. No. T80301, 1:300 dilution), Anti-N antibody (GenScript, Cat. No. T80103, 1:300 dilution), and novel coronavirus nsp12 / RdRP antibody (1:1000, (Abcam, Cat. No. ab277617).

[0051] As shown in Figure 1 , all the corresponding target bands can be detected in the monoclonal cells carrying pCI-TV-RNR1 and pCI-TV-RNR2, while the monoclonal cells carrying pCI-TV-RNR3 can only detect the target band for the novel coronavirus nsp12 / RdRP antibody, and the monoclonal cells carrying pCI-neo do not detect the target band. Therefore, the antigen protein expression of the monoclonal cells carrying pCI-TV-RNR1 and pCI-TV-RNR2 is normal, while the antigen protein expressed by the monoclonal cells carrying pCI-TV-RNR3 may only have affinity for the novel coronavirus nsp12 / RdRP antibody or its affinity for Anti-Spike antibody or Anti-N antibody decreases, or it does not have immunogenicity for the novel coronavirus spike protein and N protein.

[0052] 4. Immunogenicity experiment

[0053] (1) Test sample liquid

[0054] A large amount of recombinant plasmid pCI-TV-RNR1, pCI-TV-RNR2 or pCI-TV-RNR3 was extracted from the monoclonal cells carrying pCI-TV-RNR1, pCI-TV-RNR2 or pCI-TV-RNR3. The recombinant plasmids pCI-TV-RNR1, pCI-TV-RNR2, pCI-TV-RNR3 and pCI-neo plasmid (as a control group) were dissolved in PBS buffer solution with pH 7.4, respectively, and mixed with 3.4% polyvinylpyrrolidone aqueous solution (PVP, molecular weight 40000; Sigma-Aldrich, Cat. No. PVP40), to prepare sample liquid 1 containing 2 mg / mL pCI-TV-RNR1, sample liquid 2 containing 2 mg / mL pCI-TV-RNR2, sample liquid 3 containing 2 mg / mL pCI-TV-RNR3, and sample liquid 4 containing 2 mg / mL pCI-neo, respectively.

[0055] (2) Group immunization

[0056] Six-week-old female C57BL / 6Jlac mice (Saiye (Suzhou) Biotechnology Co., Ltd.) were randomly divided into experimental and control groups.

[0057] As shown in Figure 2 , in the experimental group, 200 μL of sample liquid 1, sample liquid 2 or sample liquid 3 was injected into the bilateral quadriceps of the mice. In the control group, 200 μL of sample liquid 4 was injected into the bilateral quadriceps of the mice. The mice were immunized by intramuscular injection on day 0, and boosted on day 14. The experimental and control group mice were injected once every two weeks, for a total of two injections. On day 28, some mice were euthanized to check for potential tissue damage. Serum samples were collected from the experimental and control groups on days 0, 14 and 28, and stored at -80°C.

[0058] (3) ELISA method for detecting three IgG antibody levels in serum

[0059] The recombinant purified S1 spike protein (HY-P78280, MCE), N protein (ab273530, abeam) and RdRP protein (DAGC202, Creative Diagnostics) were coated on the enzyme-labeled plate at 10 μg / ml. The immune serum of each group of mice on day 28 was diluted with 1% BSA PBS at 1:64, and then diluted 8 times with 2 times dilution. 100 μL of diluted detection sample was added to each well, and negative control (diluent) was added. Incubate at 37°C for hours, discard the sample, add 300 μL of washing solution to each well, soak for 1-2 minutes, then shake off, repeat 3 times, and pat dry. Dilute the HRP-conjugated anti-human IgG or IgM antibody with diluent to the appropriate concentration (e.g. 1:1000), add 100 μL of diluted enzyme-labeled secondary antibody to each well, and incubate at 37°C for 1 hour. Discard the enzyme-labeled secondary antibody, add 300 μL of washing solution to each well, soak for 1-2 minutes, then shake off, repeat 3 times, and pat dry. Add 100 μL of TMB color developing solution to each well. Incubate at room temperature for 10-30 minutes in the dark, and observe the color development. Add 50 μL of stop solution (2 mol / L H2SO4) to each well to stop the reaction. Use the enzyme-labeled instrument to read the absorbance difference between each well and the negative control group at 450 nm wavelength. The area under the curve between the lowest dilution and the highest dilution of each sample is calculated by integration, which reflects the overall reaction intensity of the sample in the entire dilution range, i.e. "dilution dependent AUC".

[0060] As shown in Figure 3 The pCI-TV-RNR1 and pCI-TV-RNR2 can produce S1 spike protein serum IgG antibodies, N protein serum IgG antibodies and RdRP protein serum IgG antibodies in the serum of mice immunized with sample liquid 1, sample liquid 2, sample liquid 3 and sample liquid 4 on day 28, respectively, while pCI-TV-RNR3 can only produce serum IgG antibodies against RdRP protein. Moreover, the S1 spike protein serum IgG antibodies, N protein serum IgG antibodies and RdRP protein serum IgG antibodies produced by pCI-TV-RNR2 are all lower than those produced by pCI-TV-RNR1.

[0061] Therefore, it is illustrated that the recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2 provided by the application can produce serum IgG antibodies against S1 spike protein, N protein and RdRP protein of the novel coronavirus, and have stronger protective effect. The recombinant plasmid pCI-TV-RNR3 can only detect serum IgG antibodies against RdRP protein in serum at day 28, and cannot detect serum IgG antibodies against N protein and trace amounts of serum IgG antibodies against S1 spike protein, and cannot produce protective effect obviously inferior to the recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2. Therefore, it is suggested that the recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2 can both develop into vaccines for the novel coronavirus, and the protective effect of the recombinant plasmid pCI-TV-RNR2 is stronger.

[0062] The pCI-TV-RNR1, pCI-TV-RNR2 or pCI-TV-RNR3 plasmid respectively carries the target sequence 1, the target sequence 2 and the target sequence 3, and it is illustrated that the pCI-TV-RNR3 plasmid carrying the target sequence 3 can only express antigens against serum IgG antibodies against RdRP protein, and antigens against serum IgG antibodies against N protein and trace amounts of serum IgG antibodies against S1 spike protein can be inhibited or cannot be correctly expressed in the fusion expression of the trivalent antigen.

[0063] (4) Enzyme-linked immunospot assay

[0064] Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of the immunized 28-day-old mice of the monoclonal cells carrying pCI-TV-RNR1, pCI-TV-RNR2, pCI-TV-RNR3 and pCI-neo respectively, and resuspended in RPMI 1640 culture medium containing 2% FBS, and the cell concentration was adjusted to 2×10 6 / mL, and used as the test sample. LEGENDplex™ Mouse Th1 / Th2 Panel (8-plex) kit (Cytelligen, item number K019-H147) was used to detect the T cell immune response of the isolated test sample.

[0065] 1) Group stimulation

[0066] RPMI 1640 culture solution containing 2% FBS containing 10 μg / mL of S1 spike protein, 10 μg / mL of N protein and 10 μg / mL of RdRP protein was prepared as a stimulation solution. 100 μL of the test sample was added to each well, 100 μL of the stimulation solution was added to each well, and incubated for 12 hours. The supernatant was separated and collected, and used as the test sample supernatant.

[0067] 2) Detection

[0068] Mixing of capture antibody with sample supernatant: Take an appropriate amount of capture antibody immobilized beads from the kit and add them to the sample supernatant to be tested. Gently vortex the mixture to ensure uniform dispersion of the beads. The capture antibodies coupled to the beads can specifically bind to the target cytokines in the sample.

[0069] Incubation: Incubate the mixture at room temperature for 2 hours in the dark to allow the capture antibodies to fully bind to the cytokines, forming antigen-antibody complexes.

[0070] Centrifugation and washing: After incubation, centrifuge the mixture to precipitate the beads and discard the supernatant. Add washing buffer, gently shake or vortex, then centrifuge again and discard the supernatant to remove unbound impurities and excess reagents. This step is usually repeated 1-2 times to ensure the surface of the beads is clean and reduce non-specific binding.

[0071] Addition of detection antibody: Add the biotinylated detection antibody mixture to the washed beads, gently vortex the mixture, and incubate at room temperature for about 1 hour in the dark. The detection antibodies can bind to the cytokines already bound to the beads, forming a "sandwich" structure.

[0072] Addition of SA-PE: After incubating the detection antibodies, directly add streptavidin-phycoerythrin conjugate (SA-PE). SA-PE can specifically bind to the biotin on the detection antibodies, allowing the complex to be labeled with phycoerythrin fluorescence. Incubate the mixture at room temperature for about 30 minutes in the dark.

[0073] Centrifugation and washing again: After incubating the SA-PE, repeat the centrifugation and washing steps to remove unbound SA-PE and other impurities.

[0074] Resuspension and detection: Resuspend the beads with an appropriate amount of detection buffer, then transfer them to the detection tube of the flow cytometer for flow cytometry detection. The flow cytometer will analyze the expression levels of various cytokines in the sample based on the intensity of the fluorescence signal on the beads.

[0075] Data analysis: Process the flow cytometry data using LEGENDplex online or offline analysis software to draw standard curves and calculate the concentrations of cytokines IFN-γ, TNF-α, IL-2, IL-6, IL-4, and IL-10 in the sample to be tested.

[0076] As Figure 4As shown, the test cell 1 and the test cell 2 can both secrete cytokines IFN-γ, TNF-α, IL-2, IL-6, IL-4 and IL-10 relative to the test cell 4, the test cell 3 can only secrete IL-2, IL-6 and IL-10 relative to the test cell 4, and the test cell 2 secretes cytokines IFN-γ, TNF-α, IL-2, IL-6, IL-4 and IL-10 at a concentration significantly higher than that of the test cell 1. The test cell 1 is peripheral blood mononuclear cells produced by mice immunized with the recombinant plasmid pCI-TV-RNR1, the test cell 2 is peripheral blood mononuclear cells produced by mice immunized with the recombinant plasmid pCI-TV-RNR2, and the test cell 3 is peripheral blood mononuclear cells produced by mice immunized with the recombinant plasmid pCI-TV-RNR3, and the test cell 4 is peripheral blood mononuclear cells produced by mice immunized with the pCI-neo plasmid.

[0077] Therefore, it is illustrated that the mice immunized with the recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2 provided by the application can produce T cells secreting multiple cytokines, and the T cells of the immunization product of the recombinant plasmid pCI-TV-RNR2 secrete cytokines at a higher concentration. T cell immunity can recognize and eliminate pathogens, helping the body to recover health. Therefore, it is suggested that the recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2 can both develop into a new coronavirus vaccine, and the recombinant plasmid pCI-TV-RNR2 has a stronger protective effect, which has a stronger T cell immune protective effect.

[0078] Safety evaluation

[0079] The liver, lung, heart, kidney, brain and spleen tissues of the mice immunized for 28 days in the experimental group and the control group were taken, sliced, and HE stained. The results are shown in Figure 5 As shown, the mice immunized with the plasmids provided by the experimental group and the control group both have no pathological changes in the liver, lung, heart, kidney, brain and spleen, etc. of the mice, and have very high safety.

[0080] Immunoprotective experiment

[0081] The serum of the mice immunized for 28 days in the experimental group and the control group was diluted by 1:16, then diluted by 2 times to 1:256, and then mixed with 200 CCID50 (20 LD 50) were mixed at a volume ratio of 1:1, a negative control was set up, and after incubation at 37°C for 2 h, 100 μL was inoculated into six-week-old female C57BL / 6Jlac mice, 4-5 mice per dilution. Before the death of all mice in the negative control group, the body weight and physiological state of the infected mice were observed and recorded. The dilution factor of the highest immune serum of the healthy mouse was used as the protective titer of the immune serum. The area under the curve between the lowest dilution and the highest dilution of each sample was calculated by integration, which reflects the overall reaction intensity of the sample in the entire dilution range, that is, "dilution dependent AUC". The neutralizing antibody production level of each group of mice was detected by animal level challenge.

[0082] As shown in Figure 6 , the animal level challenge experiment showed that the recombinant plasmids pCI-TV-RNR1, pCI-TV-RNR2 or pCI-TV-RNR3 could produce obvious neutralizing antibodies. Moreover, the neutralizing antibodies produced by the recombinant plasmid pCI-TV-RNR2 were significantly higher than those of pCI-TV-RNR1 and pCI-TV-RNR3, and pCI-neo did not produce obvious neutralizing antibodies. Therefore, it is shown that the recombinant plasmid pCI-TV-RNR2 provided by the present application has the strongest protective effect on the infected mice.

[0083] In addition, the lung tissues of the mice immunized for 28 days in the above experimental groups, control groups and negative groups (non-immunized) were taken, all RNAs were extracted, and RT-PCR detection was performed (establishment of a novel coronavirus subgenomic RNA detection method and performance evaluation; Zhao Zhiwei et al.; Journal of Practical Medicine; 2024). As shown in Figure 7 , the serum of the mice immunized with the recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2 can almost kill and clear the novel coronavirus genomic RNA in the lung tissues of the novel coronavirus virus infected mice, while the recombinant plasmids pCI-TV-RNR3 and pCI-neo still detect obvious novel coronavirus genomic RNA relative to the non-immunized group. Therefore, it is shown that the recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2 provided by the present application can produce sufficient immune protection effect to kill and clear the novel coronavirus in the lungs of mice, and the clearance effect of the mice immunized with the recombinant plasmid pCI-TV-RNR2 is the strongest.

[0084] By observing the body weight of each group of mice, the serum of the mice immunized with the recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2 can make the body weight of the novel coronavirus infected mice not decrease obviously, while the body weight of the mice immunized with the pCI-TV-RNR3 plasmid and the pCI-neo plasmid decreases and eventually dies.

[0085] The above description is only the preferred embodiment of the present application, but the protection scope of the present application is not limited to this. Any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered in the protection scope of the present application.

Claims

1. A nucleic acid, characterized in that, As shown in SEQ ID NO: 1 or 2.

2. A recombinant plasmid, characterized by, Carrying the nucleotide sequence as shown in SEQ ID NO: 1 or 2.

3. The recombinant plasmid according to claim 2, characterized in that, The recombinant plasmid is pCI-neo plasmid carrying the nucleotide sequence as shown in SEQ ID NO: 1 or 2.

4. The method for preparing a recombinant plasmid according to claim 2 or 3, wherein the DNA fragment is obtained by digesting the DNA fragment of claim 1 with a restriction enzyme. Comprise: The target fragment as shown in SEQ ID NO: 1 or 2 is connected with linearized pCI-neo; The fragment is transformed into E. coli; Positive clones are screened from the transformants; The pCI-neo plasmid carrying the sequence as shown in SEQ ID NO: 1 or 2 is extracted from the positive clones.

5. The production method according to claim 4, characterized by, The step of connecting the target fragment as shown in SEQ ID NO: 1 or 2 with linearized pCI-neo comprises: PCR amplification obtains the target fragment as shown in SEQ ID NO: 1 or 2 carrying the NheI enzyme cutting site and the SalI enzyme cutting site end; The pCI-neo plasmid is cut by NhoI and SalI enzymes respectively to obtain linearized pCI-neo; The target fragment as shown in SEQ ID NO: 1 or 2 carrying the NheI enzyme cutting site and the SalI enzyme cutting site end is connected with linearized pCI-neo respectively.

6. The preparation method according to claim 5, characterized in that, The preparation process of linearized pCI-neo fragment comprises: Take 200 ng pCI-neo plasmid, 2 μL 10 U / μL NhoI, M-buffer 10 μL and deionized water 38 μL, mix uniformly, react in 37°C water bath for 120 min, and then purify; Take 45 μL purified NhoI enzyme cut pCI-neo fragment, 10× buffer 10 μL, 1.7 mM dNTP 8 μL, 1 μL Taq DNA polymerase and deionized water 36 μL, react in 37°C water bath for 60 min, purify, and obtain the intermediate product; Take 48 μL intermediate product, 10 μL 10× Hbuffer, Sal I 2 μL and deionized water 40 μL, react in 37°C water bath for 120 min, purify, electrophorese and recover the gel to obtain linearized pCI-neo fragment.

7. The preparation method according to claim 5, characterized in that, The connection reaction step comprises: The target fragment as shown in SEQ ID NO: 1 or 2 carrying the NheI enzyme cutting site and the SalI enzyme cutting site end and linearized pCI-neo fragment are mixed in a molar ratio of 5:1, and then connected with ligation I connection enzyme in 12 μL reaction system at 16°C for 30 min, and the connection product is transformed into competent E. coli Top10, the transformed competent cells are inoculated on the surface of LB agar plate containing ampicillin, and colonies appear after incubation at 37°C overnight; Randomly pick single colonies with toothpicks and respectively transfer them into 12 LB culture mediums containing 2 mL ampicillin, and incubate at 37°C overnight with shaking; The positive bacteria liquid 100 μL is added into the flask containing 50 mL of LB medium with ampicillin, and is shaken at 30℃. After large-scale extraction and purification, the purity and concentration are measured by UV instrument, and the product is stored at -20℃ for standby. The plasmid is extracted from the bacteria liquid, and the recombinant plasmid is screened by double enzyme digestion with restriction endonuclease NheI and SalI.

8. A novel coronavirus trivalent DNA vaccine, which takes the pCI-neo plasmid shown as SEQ ID NO: 1 or 2 as an active ingredient.

9. The novel coronavirus trivalent DNA vaccine according to claim 8, further comprising 1-3.4% of a polyvinylpyrrolidone aqueous solution.

10. Use of the nucleic acid according to claim 1 or the recombinant plasmid according to any one of claims 2-3 in the preparation of a novel coronavirus vaccine.

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