Novel coronavirus trivalent DNA vaccine and preparation method thereof

Abstract

The invention relates to the technical field of novel coronavirus, in particular to a novel coronavirus trivalent DNA vaccine and a preparation method thereof. The trivalent DNA vaccine takes a recombinant plasmid carrying a nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient. The trivalent DNA vaccine provided by the invention can express a fusion antigen protein of three antigen genes of the novel coronavirus in eukaryotic cells and generate a serum IgG antibody aiming at three antigens of the novel coronavirus. In addition, the novel coronavirus trivalent DNA vaccine provided by the invention can be used for immunizing mice to generate T cells capable of secreting various cell factors, and has a stronger T cell immune protection effect.

Description

Technical Field

[0001] The present invention relates to the technical field of novel coronavirus, and specifically to a novel coronavirus trivalent DNA vaccine and a preparation method thereof. Background Art

[0002] The new coronavirus is a single-stranded positive-strand RNA virus with an envelope, encoding 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, which exists in the viral envelope as a homotrimer, consists of two functional subunits, S1 and S2. 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 connecting region, a heptad repeat sequence, 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 cuts the S1 / S2 cleavage site, the S1 subunit falls off, and the protruding FP after the S2 subunit conformation changes for membrane fusion. Studies have shown that the RBD region of the S protein of the new coronavirus is immunogenic and is the target of 90% of neutralizing antibodies in immune serum.

[0003] In order to obtain more comprehensive protection against the new coronavirus, multiple antigen proteins can often be fused and expressed as multivalent vaccines such as fusion protein vaccines and fusion DNA vaccines to obtain multiple protections in one shot. However, fusion expression does not mean that multiple antibodies or cross-protective immune effects can be induced. Fusion protein expression may interfere with each other, or multiple antigen immune effects may interfere with each other, thereby reducing or inhibiting the immune effect of one or several antigens, and thus failing to achieve the effect of multiple protections in one shot. Summary of the invention

[0004] In order to solve or partially solve the problems existing in the related art, the present invention applies to provide a new coronavirus trivalent DNA vaccine and a preparation method.

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

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

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

[0008] The third aspect of the present invention 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, transforming the connected fragment into Escherichia coli, screening positive clones from the transformants, and extracting the pCI-neo plasmid carrying SEQ ID NO: 1 or 2 from the positive clones.

[0009] In certain embodiments, the step of connecting the target fragment as shown in SEQ ID NO: 1 or 2 with the linearized pCI-neo includes: PCR amplification to obtain the target fragment as shown in SEQ ID NO: 1 or 2 carrying the NheI restriction site and the SalI restriction site at the end; using NhoI and SalI enzymes to digest the pCI-neo plasmid respectively to obtain linearized pCI-neo; and connecting the target fragment as shown in SEQ ID NO: 1 or 2 carrying the NheI restriction site and the SalI restriction site at the end with the linearized pCI-neo.

[0010] In certain embodiments, the preparation process of the linearized pCI-neo fragment includes: 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 them evenly, reacting in a 37°C water bath for 120 min and then purifying; taking 45 μL of purified NhoI-digested 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°C water bath for 60 min and purifying to obtain 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°C water bath for 120 min, purifying, electrophoresing and gel recovery to obtain a linearized pCI-neo fragment.

[0011] In certain embodiments, the ligation reaction step includes: linearizing the target fragment as shown in SEQ ID NO: 1 or 2 carrying the NheI restriction site and the SalI restriction site at the end and pCI-neo fragment at a molar ratio of 5:1, performing directional ligation with ligation I ligase in a 12 μL reaction system at 16°C for 30 minutes, transforming the ligation product into competent Escherichia coli Top10, inoculating the transformed competent cells on the surface of LB agar plates containing ampicillin, and culturing at 37°C overnight to produce colonies; randomly selecting single colonies with a toothpick and transferring them into 12 LB culture media containing 2 mL of ampicillin, and culturing at 37°C overnight with shaking; adding 100 μL of the screened positive bacterial solution to a flask containing 50 mL of LB culture media containing ampicillin and shaking at 30°C, extracting and purifying on a large scale, and UV The purity and concentration were measured by an instrument and stored at -20°C for future use; plasmids were extracted from the bacterial solution and the recombinant plasmids were screened out by double digestion with restriction endonucleases NheI and SalI.

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

[0013] In certain embodiments, the novel coronavirus trivalent DNA vaccine also includes a 1-3.4% polyvinyl pyrrolidone aqueous solution.

[0014] The fifth aspect of the present invention provides the use of the nucleic acid of the first aspect and the recombinant plasmid of the second aspect in the preparation of a new coronavirus vaccine.

[0015] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention.

[0016] Beneficial technical effects of the present invention: The present invention selects special target sequences from the novel coronavirus genome to synthesize target sequences 1, 2, and 3, and clones these target sequences into the eukaryotic expression plasmid pCI-neo, and then expresses the fusion antigen protein of the three antigen genes in Vero cells. The results show that the eukaryotic expression plasmid pCI-neo carrying target sequence 1 and target sequence 2 can express fusion proteins that can be detected by Anti-Spike antibody, Anti-N antibody and nsp12 / RdRP antibody in Vero cells, indicating that it has immunogenicity against multiple antigens of the novel coronavirus spike protein, N protein and RdRP protein. The eukaryotic expression plasmid pCI-neo carrying target sequence 3 expresses a fusion protein that can only be detected by nsp12 / RdRP antibody in Vero cells. This indicates that the antigen protein expression of monoclonal cells carrying pCI-TV-RNR1 and pCI-TV-RNR2 is normal, while the antigen protein expressed by monoclonal cells carrying pCI-TV-RNR3 may only have affinity for nsp12 / RdRP antibodies or its affinity for Anti-Spike antibodies or Anti-N antibodies is reduced, or it has no immunogenicity against the novel coronavirus spike protein and N protein.

[0017] In addition, the present invention also immunizes mice with eukaryotic expression plasmid pCI-neo carrying target sequence 1, target sequence 2, and target sequence 3. The results show that the recombinant plasmids carrying target sequence 1 and target sequence 2 can produce serum IgG antibodies against the S1 spike protein, N protein or RdRP protein of the new coronavirus, and have a stronger protective effect. However, the recombinant plasmid carrying target sequence 3 only detected RdRP protein serum IgG antibodies in the serum on the 28th day, but serum N protein serum IgG antibodies and trace amounts of S1 spike protein serum IgG antibodies could not be detected, and its protective effect was obviously inferior to that of recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2. This suggests that both recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2 can be developed into new coronavirus vaccines, and the protective effect of recombinant plasmid pCI-TV-RNR2 is stronger.

[0018] In addition, the mice immunized with recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2 provided by the present invention can produce T cells that secrete multiple cytokines, and the T cells of the recombinant plasmid pCI-TV-RNR2 immune product have a higher concentration of secreted cytokines. T cell immunity can identify and eliminate pathogens and help the body recover health. This suggests that both recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2 can be developed into new coronavirus vaccines, and the protective effect of recombinant plasmid pCI-TV-RNR2 is stronger, and it has a stronger T cell immune protection effect.

[0019] In addition, the present invention has found through testing that the recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2 provided by the present invention can produce an immune protection effect in the lungs of mice to fully kill and eliminate the new coronavirus, and the recombinant plasmid pCI-TV-RNR2 has the strongest clearance effect on mice immunized with it. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of WB detection of the fusion proteins expressed in Vero cells by pCI-neo, pCI-TV-RNR1, pCI-TV-RNR2, and pCI-TV-RNR3 plasmids, respectively, using serum IgG antibodies against the S1 spike protein, N protein, or RdRP protein of the new coronavirus.

[0021] Figure 2 This is a flow chart for immunizing mice with sample solutions containing pCI-neo, pCI-TV-RNR1, pCI-TV-RNR2, and pCI-TV-RNR3 plasmids.

[0022] Figure 3 These are the results of the serum IgG antibody level test against the S1 spike protein, N protein or RdRP protein of the new coronavirus in the serum of mice after immunizing them with sample solutions containing pCI-neo, pCI-TV-RNR1, pCI-TV-RNR2, and pCI-TV-RNR3 plasmids.

[0023] Figure 4 This is a quantitative analysis result of cytokine secretion (IFN-γ, TNF-α, IL-2, IL-6, IL-4 and IL-10) by T cells in peripheral blood mononuclear cells after mice were immunized with sample solutions containing pCI-neo, pCI-TV-RNR1, pCI-TV-RNR2, and pCI-TV-RNR3 plasmids.

[0024] Figure 5These are HE staining images of the liver, lung, heart, kidney, brain and spleen tissues of mice immunized with sample solutions containing pCI-neo, pCI-TV-RNR1, pCI-TV-RNR2 and pCI-TV-RNR3 plasmids.

[0025] Figure 6 These are the results of the neutralizing antibody level test in the animal level challenge experiment of the serum of mice immunized on the 28th day with sample solutions containing pCI-neo, pCI-TV-RNR1, pCI-TV-RNR2, and pCI-TV-RNR3 plasmids.

[0026] Figure 7 The results of the detection of the copy number of the new coronavirus genome RNA in the lung tissue of the animal level challenge experiment using the serum of mice immunized on the 28th day with sample solutions containing pCI-neo, pCI-TV-RNR1, pCI-TV-RNR2, and pCI-TV-RNR3 plasmids. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The reagents not described separately in detail in the present invention are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0028] 1. Purpose fragment 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.

[0029] 2. Recombinant plasmid In one embodiment, F1 and R1 are used as primer pairs to amplify the target sequence 1 to obtain the target fragment 1, the target fragment 1 is connected to the linearized pCI-neo fragment, the connected fragment is transformed into Escherichia coli, positive clones are screened from the transformants, and the pCI-neo recombinant plasmid carrying the target sequence 1 is extracted from the positive clones. Among them, F1 is: ccgGCTAGCagagtccaaccaacagaatctattgttagatttc, which is the recognition sequence of the NheI enzyme in capital letters, SEQ ID NO: 4. R1 is: cgGTCGACtttaactagcattgtatgttgagagcaaaattcatgaggtcc, which is the recognition sequence of the SalI enzyme in capital letters, SEQ ID NO: 5.

[0030] In one embodiment, F2 and R2 are used as primer pairs to amplify the target sequence 2 to obtain the target fragment 2, the target fragment 2 is connected to the linearized pCI-neo fragment, the connected fragment is transformed into Escherichia coli, positive clones are screened from the transformants, and the pCI-neo recombinant plasmid carrying the target sequence 2 is extracted from the positive clones. Among them, F2 is: ccgGCTAGCtctaacaatcttgattctaaggttggtgg, which is the recognition sequence of the NheI enzyme in capital letters, SEQ ID NO: 6. R2 is: cgGTCGACtttaactagcattgtatgttgagagcaaaattcatgaggtcc, which is the recognition sequence of the SalI enzyme in capital letters, SEQ ID NO: 7.

[0031] In one embodiment, F3 and R3 are used as primer pairs to amplify the target sequence 3 to obtain the target fragment 3, the target fragment 3 is connected to the linearized pCI-neo fragment, the connected fragment is transformed into Escherichia coli, positive clones are screened from the transformants, and the pCI-neo recombinant plasmid carrying the target sequence 3 is extracted from the positive clones. Among them, F3 is: ccgGCTAGCagagtccaaccaacagaatctattgttagatttc, which is the recognition sequence of the NheI enzyme in capital letters, SEQ ID NO: 8. R3 is: cgGTCGACggcccctaggattcttgatggatctgggtaaggaaggtacacataa, which is the recognition sequence of the SalI enzyme in capital letters, SEQ ID NO: 9.

[0032] The reaction system for PCR amplification was: 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 enzyme-free double distilled water was used to make up to 50 μL. The reaction conditions were: 95°C for 5 min; 95°C for 40 s, 57°C for 40 s, 72°C for 1.5 min, 35 cycles; 72°C for 10 min, and the reaction was terminated at 4°C.

[0033] The target fragments 1, 2 and 3 can be obtained by using other primers or chemical synthesis methods, and are not limited to the steps in the above embodiment.

[0034] The preparation process of the linearized pCI-neo fragment is as follows: Take 200ng pCI-neo plasmid (Promega), 2μL 10 U / μL NhoI (ER0971, ThermoScientific TM), M-buffer 10μL and deionized water 38μL were mixed evenly, reacted in a 37℃ water bath for 120min, and purified strictly according to the Cycle-pure Kit (Omega Bio-tek) operation manual. Then, 45μL of purified NhoI-digested pCI-neo fragment, 10μL of 10× buffer, 8μL of 1.7mM dNTP, 1μL of Taq DNA polymerase and 36μL of deionized water were taken, reacted in a 37℃ water bath for 60 min to purify the intermediate product. Take 48μL of the intermediate product, 10μL of 10×Hbuffer, 2μL of Sal I and 40μL of deionized water, reacted in a 37℃ water bath for 120 min. Purification, electrophoresis and gel recovery were performed to obtain the linearized pCI-neo fragment.

[0035] The steps of connecting the target fragment 1, target fragment 2 or target fragment 3 to the linearized pCI-neo fragment are: Linearize the pCI-neo fragments of target fragment 1, target fragment 2 or target fragment 3 at a molar ratio of 5:1, and perform directional ligation with ligation I ligase in a 12μL reaction system at 16℃ for 30min. Then transform the ligation products into competent Escherichia coli Top10. The transformed competent cells are inoculated on the surface of LB agar plates containing ampicillin. Colonies appear after culturing at 37°C overnight. Use a toothpick to randomly select single colonies and transfer them to 12 LB culture media containing 2mL of ampicillin, and culture them at 37°C with shaking (200 rpm) overnight. Add 100μL of the screened positive bacterial solution to a flask containing 50mL of LB culture media containing ampicillin and shake at 30℃ overnight. Large-scale extraction and purification. Measure its purity and concentration with a UV instrument and store at -20℃ for future use. Plasmids were 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 was screened out by double digestion with restriction endonucleases NheI and SalI. TM The plasmid DNA extraction kit and purification kit were purchased from Sigma, USA.

[0036] 3. Eukaryotic expression of target sequences (1) Linearization of recombinant plasmid PCI-neo plasmid was linearized by restriction endonuclease BamH I to facilitate transfection: PCI-neo-mdr1 100μL, buffer 15μL, BamH I 6μL and deionized water 29μL were mixed evenly and reacted in a 37℃ water bath for 120 min. Purification was the same as above. Purification and concentration were measured on a UV instrument.

[0037] (2) Vero cell recovery and culture Quickly take out the Vero cell cryopreservation tube (Cat. No.: CL-0242, Wuhan Punosai) from the liquid nitrogen, put it into a 37℃ water bath and shake it to completely dissolve it within 1 min, transfer the cell suspension to a centrifuge tube, centrifuge at 1000r / min for 3 min, discard the supernatant, add 3mL DMEM to completely dissolve the cells, and transfer the suspension to a 25cm 3 Add about 10 mL of RPMI-1640 complete medium to a glass bottle and incubate at 37°C and 5% CO 2 Culture in an incubator and change the medium every 2-3 days. Vero cells are adherent cells. When the cells fill the glass bottle, digest and subculture.

[0038] (3) Transfection of Vero cells Inoculate cells 1 day before transfection, and the cells should reach 60-80% coverage on the day of transfection. Generally, 6-well culture dishes, 2 mL per well, culture 3×10 5Cells. On the day of transfection, replace the medium with fresh serum-free medium within 2 hours before adding the liposome / DNA mixture. Prepare the liposome / DNA mixture. Solution A: Prepare 2.5 μg of linearized PCI-neo recombinant plasmid (carrying target sequence 1, 2, or 3, respectively) with 20 mM HEPES (pH 7.4) to a total volume of 25 μL. Solution B: Dilute 15 μL of lipofectamine with 20 mM HEPES (pH 7.4) to a total volume of 35 μL. Gently mix solutions A and B (pipette gently several times, do not shake) and incubate at room temperature for 15 min to allow the liposome / DNA mixture to form. Without removing the medium, add 60 μL of the liposome / DNA mixture dropwise, shaking the culture plate gently while adding. Incubate at 37°C for 6 hours. After 6 hours, replace the medium and add 2 mL of fresh growth medium containing serum. 24 h after transfection, culture medium containing G418 (400 μg / mL) was used. After 3 days, the culture medium was changed once and the G418 concentration was increased to 600 μg / mL. After 5 days, the G418 concentration was increased to 800 μg / mL. After 8 days, the surviving cells in the 6-well culture dish were transferred to culture flasks for culture with a G418 concentration of 400 μg / mL. Screening was carried out at this concentration for more than 20 days until the cells grew stably and were expanded for culture.

[0039] (4) Western blot Western blot method was used to identify the expression of target sequence 1, target sequence 2, and target sequence 3 in monoclonal cell lines. Among them, monoclonal cells were transfected with the recombinant plasmid carrying target sequence 1 (pCI-TV-RNR1), monoclonal cells were transfected with the recombinant plasmid carrying target sequence 2 (pCI-TV-RNR2), and monoclonal cells were transfected with the recombinant plasmid carrying target sequence 3 (pCI-TV-RNR3).

[0040] The 12th and 15th generation monoclonal cell lines were selected, and the screening cells that had grown monolayers were fully lysed using RIPA lysis buffer. The quantitative proteins were taken for SDS-PAGE separation of proteins of different sizes. The separated proteins were transferred to the PVDF membrane by a membrane transfer instrument. The PVDF membrane was washed 3 times with TBST buffer (pH=7.4, 2‰ Tween 20), and blocked with 4% skim milk at room temperature for 2 hours. Incubate with primary antibody at 4℃ overnight. Rinse 3 times with TBST buffer, dilute the goat anti-mouse secondary antibody labeled with horseradish peroxidase (HRP) at 1:500, and incubate at room temperature for 2 hours. Rinse 3 times with TBST buffer and observe after color development with DAB substrate. Among them, the primary antibodies are 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).

[0041] like Figure 1 As shown, 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 bands against the new coronavirus nsp12 / RdRP antibody, and the monoclonal cells carrying pCI-neo did not detect the target bands. This shows that 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 new coronavirus nsp12 / RdRP antibody or its affinity for Anti-Spike antibody or Anti-N antibody is reduced, or it does not have immunogenicity against the new coronavirus spike protein and N protein.

[0042] 4. Immunogenicity test (1) Test sample liquid A large amount of recombinant plasmid pCI-TV-RNR1, pCI-TV-RNR2 or pCI-TV-RNR3 was extracted from 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 at pH 7.4 and mixed with 3.4% polyvinylpyrrolidone aqueous solution (PVP, molecular weight 40000; Sigma-Aldrich, Cat. No. PVP40) to prepare sample solution 1 containing 2 mg / mL pCI-TV-RNR1, sample solution 2 containing 2 mg / mL pCI-TV-RNR2, sample solution 3 containing 2 mg / mL pCI-TV-RNR3, and sample solution 4 containing 2 mg / mL pCI-neo.

[0043] (2) Group immunization Six-week-old female C57BL / 6Jlac mice (Saiye (Suzhou) Biotechnology Co., Ltd.) were randomly divided into experimental and control groups.

[0044] like Figure 2 As shown, in the experimental group, 200 μL of sample solution 1, sample solution 2 or sample solution 3 were injected into the anterior thigh muscles of the mice. In the control group, 200 μL of sample solution 4 was injected into the anterior thigh muscles of the mice. The mice were immunized by intramuscular injection on day 0 and boosted on day 14. Both 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 examine potential tissue damage. Serum samples from the experimental and control groups were collected on days 0, 14, and 28 and stored at -80°C.

[0045] (3) ELISA method to detect the levels of three types of IgG antibodies in serum The ELISA plate was pre-coated with 10 µg / ml recombinant purified S1 spike protein (HY-P78280, MCE), N protein (ab273530, abcam) and RdRP protein (DAGC202, Creative Diagnostics). The immune serum of mice in each group on day 28 was diluted 1:64 with 1% BSA in PBS, and then diluted 8 times in 2-fold gradients. 100 µL of the diluted test sample was added to each well, and the negative control (diluent) was added at the same time. The plates were incubated at 37°C for 1 hour, the samples were discarded, 300 µL of washing solution was added to each well, and the plates were soaked for 1-2 minutes, then shaken off. This was repeated 3 times and patted dry. Dilute HRP-conjugated anti-human IgG or IgM antibody to an appropriate concentration (e.g. 1:1000) with diluent, add 100 µL of diluted enzyme-labeled secondary antibody to each well, incubate at 37°C for 1 hour, discard enzyme-labeled secondary antibody, add 300 µL of washing solution to each well, soak for 1-2 minutes, shake off, repeat 3 times, and pat dry. Add 100 µL of TMB colorimetric solution to each well. Incubate at room temperature in the dark for 10-30 minutes and observe the color development. Add 50 µL of stop solution (2 mol / L H 2 SO 4 ) to terminate the reaction. Use a microplate reader to read the absorbance difference between each well and the negative control group at a wavelength of 450 nm. The area under the curve from the lowest dilution to the highest dilution of each sample. It is calculated by integration and reflects the overall reaction intensity of the sample in the entire dilution range, which is the "dilution dependent AUC".

[0046] like Figure 3 As shown, in the serum of the 28th mouse immunized with sample solution 1, sample solution 2, sample solution 3 and sample solution 4, pCI-TV-RNR1 and pCI-TV-RNR2 can produce serum IgG antibodies containing S1 spike protein, N protein and RdRP protein, while pCI-TV-RNR3 can only produce serum IgG antibodies against RdRP protein. Moreover, the levels of S1 spike protein serum IgG antibodies, N protein serum IgG antibodies and RdRP protein serum IgG antibodies produced by pCI-TV-RNR2 are not as good as those of pCI-TV-RNR1.

[0047] This shows that the recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2 provided by the present invention can produce serum IgG antibodies against the S1 spike protein, N protein and RdRP protein of the new coronavirus, and have a stronger protective effect. The recombinant plasmid pCI-TV-RNR3 only detected RdRP protein serum IgG antibodies in the serum on the 28th day, but serum N protein serum IgG antibodies and trace amounts of S1 spike protein serum IgG antibodies could not be detected, and its protective effect was obviously inferior to that of the recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2. This suggests that both recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2 can be developed into new coronavirus vaccines, and the protective effect of the recombinant plasmid pCI-TV-RNR2 is stronger.

[0048] The pCI-TV-RNR1, pCI-TV-RNR2 or pCI-TV-RNR3 plasmids carry target sequence 1, target sequence 2 and target sequence 3, respectively, indicating that the pCI-TV-RNR3 plasmid carrying target sequence 3 can only express antigens against serum IgG antibodies against RdRP protein, and its antigens against serum IgG antibodies against N protein and trace amounts of serum IgG antibodies against S1 spike protein may be inhibited or cannot be expressed correctly in the fusion expression of the trivalent antigen.

[0049] (4) ELISA Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of mice immunized for 28 days with monoclonal cells carrying pCI-TV-RNR1, pCI-TV-RNR2, pCI-TV-RNR3, and pCI-neo, respectively, and resuspended in RPMI 1640 medium containing 2% FBS to adjust the cell concentration to 2 × 10 6 / mL, respectively, as the test samples. The LEGENDplex™ Mouse Th1 / Th2Panel (8-plex) kit (Cytelligen, catalog number K019-H147) was used to detect the T cell immune response of the separated test samples.

[0050] 1) Group stimulation Prepare RPMI 1640 culture medium containing 2% FBS containing 10 μg / mL S1 spike protein, 10 μg / mL N protein and 10 μg / mL RdRP protein as the stimulation solution. Add 100 μL of the sample to be tested to each well, add 100 μL of the stimulation solution to each well, incubate for 12 hours, separate and collect the supernatant, and use it as the supernatant of the sample to be tested.

[0051] 2) Detection Mix the capture antibody with the supernatant of the sample to be tested: Take an appropriate amount of capture antibody-immobilized beads from the kit, add them to the supernatant of the sample to be tested, and gently vortex to mix so that the beads are evenly dispersed. The capture antibody coupled to the beads can specifically bind to the target cytokine in the sample.

[0052] Incubation: Incubate the mixture at room temperature in the dark for 2 hours to allow the capture antibody to fully bind to the cytokine and form an antigen-antibody complex.

[0053] Centrifugation and washing: After the incubation, pellet the beads by centrifugation and discard the supernatant. Add wash 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 bead surface is clean and reduce non-specific binding.

[0054] Add detection antibody: Add the biotinylated detection antibody mixture to the washed beads, vortex gently to mix, and incubate at room temperature in the dark for about 1 hour. The detection antibody can bind to the cytokines already bound to the beads to form a "sandwich" structure.

[0055] Add SA-PE: After incubation with the detection antibody, add streptavidin-phycoerythrin conjugate (SA - PE) directly. SA-PE can specifically bind to the biotin on the detection antibody, thereby making the complex carry the phycoerythrin fluorescent label. Incubate the mixture at room temperature in the dark for about 30 minutes.

[0056] Centrifuge and wash again: After the SA-PE incubation is completed, repeat the centrifugation and washing steps to remove unbound SA-PE and other impurities.

[0057] Resuspend and detect: Resuspend the beads with an appropriate amount of detection buffer and 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 fluorescent signal on the beads.

[0058] Data analysis: Flow cytometry data were processed by 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 samples to be tested.

[0059] like Figure 4As shown, the test cells 1 and 2 can 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 concentrations significantly higher than that of the test cell 1. The test cell 1 is a peripheral blood mononuclear cell produced by mice immunized with the recombinant plasmid pCI-TV-RNR1, the test cell 2 is a peripheral blood mononuclear cell produced by mice immunized with the recombinant plasmid pCI-TV-RNR2, the test cell 3 is a peripheral blood mononuclear cell produced by mice immunized with the recombinant plasmid pCI-TV-RNR3, and the test cell 4 is a peripheral blood mononuclear cell produced by mice immunized with the pCI-neo plasmid.

[0060] This shows that the mice immunized with the recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2 provided by the present invention can produce T cells that secrete multiple cytokines, and the T cells of the recombinant plasmid pCI-TV-RNR2 immune product have a higher concentration of secreted cytokines. T cell immunity can identify and eliminate pathogens and help the body recover health. This suggests that both recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2 can be developed into new coronavirus vaccines, and the protective effect of the recombinant plasmid pCI-TV-RNR2 is stronger, and it has a stronger T cell immune protection effect.

[0061] Safety evaluation The liver, lung, heart, kidney, brain and spleen tissues of the mice in the experimental group and the control group on the 28th day of immunization were sliced ​​and stained with HE. Figure 5 As shown. Whether the plasmids provided by the experimental group or the control group immunized mice, pathological changes were produced in the liver, lungs, heart, kidneys, brain, spleen and other major organs of the mice, and the safety was very high.

[0062] Immunoprotective assay The serum from the experimental group and the control group on the 28th day of immunization was diluted 1:16, then diluted 2 times to 1:256, and then mixed with 200 CCID50 (20 LD 50) of the novel coronavirus (isolated from throat swab samples of infected persons in the People's Hospital affiliated to Hubei Medical College) were mixed in a volume ratio of 1:1, and a negative control was set up. After incubation at 37°C for 2 hours, they were inoculated into six-week-old female C57BL / 6Jlac mice, 100μL each, 4-5 mice / dilution. Under the premise that all the negative control groups died, the weight and physiological state of the challenged mice were observed and recorded. The highest dilution multiple of all healthy immune sera was taken as the mouse protection titer of the immune serum. The area under the curve from the lowest dilution to the highest dilution of each sample. It is calculated by integration, reflecting the overall reaction intensity of the sample in the entire dilution range, that is, "dilution dependentAUC", and the neutralizing antibody production level of each group of mice was detected by animal level challenge method.

[0063] like Figure 6 As shown, the challenge experiment at the animal level showed that the recombinant plasmids pCI-TV-RNR1, pCI-TV-RNR2 or pCI-TV-RNR3 can produce obvious neutralizing antibodies. Moreover, the neutralizing antibodies produced by the recombinant plasmid pCI-TV-RNR2 are significantly higher than those of pCI-TV-RNR1 and pCI-TV-RNR3, and pCI-neo does not produce obvious neutralizing antibodies. This shows that the recombinant plasmid pCI-TV-RNR2 provided by the present invention has the strongest protective effect on challenged mice.

[0064] In addition, the lung tissues of mice in the experimental group, control group, and negative group (unimmunized) on the 28th day of immunization were taken to extract all RNA for RT-PCR detection (Establishment and performance evaluation of the detection method of subgenomic RNA of new coronavirus; Zhao Zhiwei et al.; Journal of Practical Medicine; 2024). Figure 7 As shown, the serum of mice immunized with recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2 can make the lung tissue of mice challenged with the new coronavirus virus almost free of the new coronavirus genomic RNA, while the recombinant plasmids pCI-TV-RNR3 and pCI-neo still detected obvious new coronavirus genomic RNA relative to the non-immunized group. This shows that the recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2 provided by the present invention can produce an immune protection effect in the lungs of mice that fully kills and eliminates the new coronavirus, and the removal effect produced by mice immunized with the recombinant plasmid pCI-TV-RNR2 is the strongest.

[0065] By observing the weight of each group of mice, the serum of mice immunized with recombinant plasmids pCI-TV-RNR1 and pCI-TV-RNR2 could cause no significant decrease in the weight of mice challenged with the new coronavirus, while the mice immunized with pCI-TV-RNR3 plasmid and pCI-neo plasmid lost weight and eventually died.

[0066] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

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

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

3. The recombinant plasmid according to claim 2, characterized in that The recombinant plasmid is a pCI-neo plasmid carrying a 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, characterized in that: include: Connect the target fragment as shown in SEQ ID NO: 1 or 2 to the linearized pCI-neo; The ligated fragments were transformed into E. coli; Screening positive clones from transformants; The pCI-neo plasmid carrying SEQ ID NO: 1 or 2 was extracted from the positive clones.

5. The preparation method according to claim 4, characterized in that: The step of connecting the target fragment as shown in SEQ ID NO: 1 or 2 with linearized pCI-neo comprises: PCR amplification to obtain the target fragment as shown in SEQ ID NO: 1 or 2 carrying the NheI restriction site and the SalI restriction site end; The pCI-neo plasmid was digested with NhoI and SalI enzymes to obtain linearized pCI-neo; The target fragments as shown in SEQ ID NO: 1 or 2 carrying the NheI restriction site and the SalI restriction site at the end were ligated to the linearized pCI-neo.

6. The preparation method according to claim 5, characterized in that: The preparation process of linearized pCI-neo fragment includes: Take 200ng pCI-neo plasmid, 2μL 10 U / μL NhoI, 10μL M-buffer and 38μL deionized water, mix well, react in a 37℃ water bath for 120min and purify; Take 45 μL of purified NhoI-digested pCI-neo fragment, 10 μL of 10× buffer, 8 μL of 1.7 mM dNTP, 1 μL of TaqDNA polymerase and 36 μL of deionized water, react in a 37°C water bath for 60 min to purify and obtain the intermediate product; Take 48 μL of the intermediate product, 10 μL of 10× Hbuffer, 2 μL of Sal I and 40 μL of deionized water, react in a 37°C water bath for 120 min, purify, perform electrophoresis and gel recovery to obtain the linearized pCI-neo fragment.

7. The preparation method according to claim 5, characterized in that: The ligation reaction steps include: The target fragments as shown in SEQ ID NO: 1 or 2 carrying NheI restriction site and SalI restriction site ends were linearized and pCI-neo fragments were mixed at a molar ratio of 5:1, and directional ligation was performed with ligation I ligase in a 12 μL reaction system at 16°C for 30 minutes, and the ligation products were transformed into competent Escherichia coli Top10. The transformed competent cells were inoculated on the surface of LB agar plates containing ampicillin, and colonies appeared after being cultured at 37°C overnight; Use a toothpick to randomly pick a single colony and transfer it to 12 LB medium containing 2 mL of ampicillin, and culture at 37°C with shaking overnight; 100 μL of the screened positive bacterial solution was added to a 50 mL flask of LB medium containing ampicillin and shaken at 30°C for large-scale extraction and purification. The purity and concentration were measured by UV instrument and stored at -20°C for future use. The plasmid was extracted from the bacterial solution, and the recombinant plasmid was screened out by double digestion with restriction endonucleases NheI and SalI.

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

9. The novel coronavirus trivalent DNA vaccine according to claim 5, further comprising a 1-3.4% polyvinyl pyrrolidone aqueous solution.

10. Use of the nucleic acid as described in claim 1 or the recombinant plasmid as described in any one of claims 2 to 3 in the preparation of a new coronavirus vaccine.

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