Novel coronavirus pneumonia DNA nano vaccine and preparation method thereof
阅读说明：本技术 新型冠状病毒肺炎dna纳米疫苗及其制备方法 (Novel coronavirus pneumonia DNA nano vaccine and preparation method thereof ) 是由 米鹏 仝爱平 卓维玲 于 2021-07-28 设计创作，主要内容包括：本发明公开了一种针对新型冠状病毒肺炎DNA纳米疫苗及其制备方法,将传统编码新型冠状病毒外壳上刺突蛋白(S)的DNA胞质尾区域删除为S.dCT DNA,与阳离子聚合物通过静电相互作用自组装成纳米疫苗,其中阳离子聚合物为PAsp(EDA)、PAsp(DET)、PAsp(TET)、PAsp(TEP)、PAsp(BAP)、PAsp(TAE)或PAsp(TAP)中的一种或它们的同类型衍生物。本发明制备的DNA纳米疫苗有较高的转染效率,优化的DNA可以增加刺突蛋白在细胞膜上的表达水平,引起体液免疫,引发高滴度的针对假病毒的中和抗体,并且安全性高,可用于预防和控制新型冠状肺炎。(The invention discloses a DNA nano vaccine aiming at novel coronavirus pneumonia and a preparation method thereof, wherein a DNA cytoplasmic tail region of a spike protein (S) on a traditional coding novel coronavirus shell is deleted to be S.dCT DNA, and the DNA cytoplasmic tail region and a cationic polymer are self-assembled into the nano vaccine through electrostatic interaction, wherein the cationic polymer is one of or the same type of derivatives of PASp (EDA), PASp (DET), PASp (TET), PASp (TEP), PASp (BAP), PASp (TAE) or PASp (TAP). The DNA nano vaccine prepared by the invention has higher transfection efficiency, the optimized DNA can increase the expression level of spike protein on cell membranes, cause humoral immunity and initiate high-titer neutralizing antibodies aiming at pseudoviruses, and the DNA nano vaccine has high safety and can be used for preventing and controlling novel coronary pneumonia.)
1. A DNA nano-class vaccine for treating coronavirus pneumonia is prepared from the plasmid carrier containing DNA for coding the coat S protein of coronavirus and deleting 99 nucleotides from its tail region and the cationic polymer (polyaspartic acid) through electrostatic self-assembly.
2. The DNA nano-vaccine of claim 1, characterized in that the nucleotide sequence of the DNA is shown in SEQ ID No. 1.
3. The DNA nanoball of claim 1, wherein the plasmid vector is a pCAGGS vector.
4. The DNA nano-vaccine of claim 1, characterized in that the molar ratio of the protonatable nitrogen atom in the cationic polymer to the phosphate in the plasmid vector is 1-60.
5. The DNA nanoball of claim 1, wherein the cationic polymer is poly [ N- (2-aminoethyl) aspartic acid ], poly { N- [ N ' - (2-aminoethyl) -2-aminoethyl ] aspartic acid }, poly (N- { N ' - [ N "- (2-aminoethyl) -2-aminoethyl ] -2-aminoethyl } aspartic acid), poly [ N- (N ' - { N" - [ N ' "- (2-aminoethyl) -2-aminoethyl ] -2-aminoethyl } -2-aminoethyl) aspartic acid ], poly (N- { N ' - [ N" - (3-aminoethyl) -3-aminoethyl ] -3-aminoethyl } aspartic acid) ], or a mixture thereof, One of poly (N, N, N-3 (2-aminoethyl) aspartic acid) or poly (N, N, N-3 (3-aminopropyl) aspartic acid) or their derivatives of the same type, and the molecular weight is 1-40 KDa.
6. The method for preparing the novel coronavirus pneumonia DNA nano-vaccine of any one of claims 1-5, which comprises the following steps:
(1) preparing a plasmid vector and a cationic polymer into solutions with proper concentrations by using hydroxyethyl piperazine ethanethiosulfonic acid buffer solutions with the pH value of 7.40 respectively;
(2) the plasmid vector solution and the cationic polymer solution are uniformly mixed according to a proper proportion, and the mixture is stood to ensure that the cationic polymer with positive electricity and the plasmid vector with negative electricity form the DNA nano vaccine through electrostatic interaction.
7. The method for preparing the novel DNA nano-vaccine for coronavirus pneumonia of claim 6, wherein in the step (1), the concentration of the cationic polymer is 1.0-10 μ g/μ L, and the concentration of the plasmid vector is 0.1-10 μ g/μ L.
8. Use of the DNA nano-vaccine of any one of claims 1 to 5 for the preparation of a medicament for the prevention or/and treatment of novel coronavirus pneumonia.
Technical Field
The invention belongs to the field of vaccines, and particularly relates to a novel coronavirus pneumonia DNA nano vaccine and a preparation method thereof.
Background
The novel Coronavirus (SARS-CoV-2) is the cause of the outbreak of the novel Coronavirus pneumonia (COVID-19), and the outbreak and the persistence of the epidemic situation start from the end of 2019 in the world, which causes huge casualties and major economic loss. In the face of repeated and unusual epidemics and infections, the only Vaccine is the weapon that we are most effective against viruses (Vaccine39(2) (2021)197- "201).
The protein coat of SARS-CoV-2 is composed of spike protein (S), membrane glycoprotein (M), nucleocapsid protein (N), hemagglutinin esterase dimer protein (He) and envelope protein (E). The S protein is a virus fusion protein, mediates the attachment of the virus to an angiotensin converting enzyme 2(ACE2) receptor on the cell surface, performs membrane fusion, and is absorbed by endosomes after the virus enters cells. Proteolytic cleavage of the S protein and fusion of the viral membrane to the endosomal membrane triggers release of viral RNA into the cytoplasm (Nat Microbiol 5(4) (2020) 562-. Therefore, the S protein is the most important immunogenic protein of SARS-CoV-2, can stimulate the organism to produce specific neutralizing antibody and mediate the cellular immunity of the organism, and is the most key target point for developing SARS-CoV-2 vaccine.
At present, various SARS-CoV-2 candidate vaccines are under development, which is mainly divided into five technical directions: inactivated vaccine, recombinant protein vaccine, adenovirus vector vaccine, attenuated influenza virus vector vaccine and nucleic acid vaccine (also divided into mRNA vaccine and DNA vaccine). Recently, inactivated vaccines in China have been marketed (Cell 182(3) (2020)713), BNT162b1(Interim report. medRxiv; 2020.DOI:10.1101/2020.06.30.20142570.) developed by Pereri in America and Byentake in Germany, and mRNA-1273 and the like (N.Engl. J.Med.383(20) (2020) 1920-1931) developed by Mordana in America.
The DNA vaccine technology is relatively mature, plasmid DNA can exist in a host body for a long time, antigen genes are continuously expressed in the body to generate antigen proteins, an organism immune system is continuously stimulated to generate long-range immunity, the immune effect is reliable, the gene vaccine can generate humoral immune response and can lead cytotoxic T lymphocytes to be activated to induce cellular immunity, and the traditional vaccine only can induce the cellular immunity but has the danger of the virulence of the live vaccine rising. DNA vaccines are safer than inactivated vaccines that require in vitro culture of coronaviruses, and inactivated vaccines risk causing enhanced antibody-dependent infection (ADE), antibodies increase the ability of the virus to infect and ultimately worsen the disease (Nature Microbiology 5(10) (2020) 1185-1191). Compared to mRNA vaccines, DNA is more stable, temperature stable and free of cold chain limitations, which is an important advantage for delivery into resource-limited environments.
Gene delivery vectors are a key part of DNA vaccines and mainly include both viral and non-viral vectors. Although the transfection efficiency of the viral vector is high, the development and application of the viral vector are greatly limited due to difficulties in preparation and production, high immunogenicity, and limitations on the size of the entrapped gene. Non-viral vectors consist primarily of cationic polymers and cationic liposomes, which deliver genes into cells by electrostatic interaction with the genes. The cationic polymer is easy to synthesize and modify, has no immunogenicity, and can be used as a good gene delivery vector to prepare a novel DNA nano vaccine for coronavirus pneumonia, but the problems of transfection efficiency, stability and safety, particularly ADE effect avoidance and the like, need to be concerned at the same time.
Walls et al (Cell 183(5) (2020)1367) constructed plasmid expressing S protein receptor binding domain, by plasmid transfection of 293 cells, extracted and purified S protein receptor binding domain from 293 cells, prepared S protein nanoparticle vaccine, induced strong neutralizing antibody response in BALB/c mice. This technique has the following disadvantages:
(1) the purification process of extracting and purifying protein and preparing nanoparticles is more complicated, and the production cost is higher;
(2) there is no concern as to whether the vaccine is safe and will cause ADE effects.
Smith et al (Nature Communications 11(1) (2020) constructed plasmids expressing the S protein, extracted the DNA of interest, injected into the anterior tibialis muscle of mice, and then used an in vivo electroporation device to pulse the muscle with a constant current, causing the DNA to transfect into the cells of the mice, the DNA vaccine stimulates the mice to produce neutralizing antibodies, blocks the binding of the S protein to the ACE2 receptor, and prevents the infection of SARS-CoV-2 virus. This technique has the following disadvantages:
(1) the DNA sequence designed in this document is a full-length DNA sequence expressing S protein, and most of the expressed S protein is retained in endoplasmic reticulum membrane (Journal of Virology,2020.94(21)), and is less secreted to the outside of the cell;
(2) special electroporation equipment is needed to promote the transfection of DNA, so the cost is high;
(3) the conditions of electroporation are controlled, otherwise cell death rate is high or transfection efficiency is low.
Disclosure of Invention
The technical problem to be solved by the invention is as follows:
(1) aiming at the situation that the existing novel coronary pneumonia is abused frequently, a vaccine for preventing the infection of the novel coronary pneumonia is constructed, S protein is selected as a target point according to the characteristics of the virus, and DNA which can stably express the S protein and secrete the S protein to the outside of cells to be identified by immune cells so as to cause strong immune response is constructed.
(2) Provides a preparation method of a novel coronavirus pneumonia DNA nano vaccine.
(3) Provides the application of the novel coronavirus pneumonia DNA nano-vaccine.
The technical scheme of the invention is as follows: a DNA nano-class vaccine for treating coronavirus pneumonia is prepared from the plasmid carrier containing DNA for coding the coat S protein of coronavirus and deleting 99 nucleotides from its tail region and the cationic polymer (polyaspartic acid) through electrostatic self-assembly.
Further, the nucleotide sequence of the DNA encoding the novel coronavirus coat S protein from which 99 nucleotides of the cytoplasmic tail region were deleted is shown in SEQ ID No. 1.
Further, the plasmid vector is a pCAGGS vector.
Further, the mole ratio of protonatable nitrogen atoms in the cationic polymer to phosphate groups in the plasmid vector is 1-60.
Further, the cationic polymer poly [ N- (2-aminoethyl) aspartic acid ] is abbreviated as PASp (EDA) and has a molecular weight of 1.0-40 KDa; poly { N- [ N' - (2-aminoethyl) -2-aminoethyl ] aspartic acid } abbreviated as PASp (DET) and having a molecular weight of 1.0-40 KDa; poly (N- { N' - [ N "- (2-aminoethyl) -2-aminoethyl ] -2-aminoethyl } aspartic acid), abbreviated as PASp (TET), has a molecular weight of 1.0-40 KDa; poly [ N- (N '- { N "- [ N"' - (2-aminoethyl) -2-aminoethyl ] -2-aminoethyl } -2-aminoethyl) aspartic acid ] abbreviated as pasp (tep) and having a molecular weight of 1.0-40 KDa; poly (N- { N '- [ N' - (3-aminoethyl) -3-aminoethyl ] -3-aminoethyl } aspartic acid), abbreviated as PASp (BAP), has a molecular weight of 1.0 to 40 kDa; poly (N, N, N-3 (2-aminoethyl) aspartic acid), abbreviated as PASp (TAE), has a molecular weight of 1.0 to 40 kDa; or poly (N, N, N-3 (3-aminopropyl) aspartic acid) abbreviated as PASP (TAP) and having a molecular weight of 1.0 to 40 kDa.
The preparation method of the novel coronavirus pneumonia DNA nano vaccine comprises the following steps:
(1) plasmid vector and cationic polymer were prepared into appropriate concentration solutions using hydroxyethylpiperazine ethanethiosulfonic acid (HEPES) buffer solution of pH 7.40, respectively.
(2) The plasmid carrier solution and the cationic polymer solution are mixed evenly according to a proper proportion, and the mixture is stood to lead the cationic polymer with positive electricity and the DNA with negative electricity to form the DNA nano vaccine through electrostatic interaction, wherein the grain diameter of the DNA nano vaccine is 40-100 nm.
Further, the concentration of the cationic polymer is 1.0-10 mug/muL, and the concentration of the plasmid vector is 0.1-10 mug/muL; the molar ratio of the protonatable nitrogen atoms in the cationic polymer to the phosphate groups in the plasmid vector (N/P ratio) is from 1 to 60.
The DNA nano vaccine of the invention is applied to the preparation of the medicine for preventing or/and treating the novel coronavirus pneumonia.
Compared with the prior art, the invention has the following beneficial effects:
(1) the novel coronavirus DNA nano vaccine gene vector provided by the invention is a cationic polymer, has low toxicity and biodegradability, and improves the safety and stability of the vaccine.
(2) The novel coronavirus DNA nano-vaccine provided by the invention aims at the S protein of a novel coronavirus, and the DNA of the S protein is deleted in a cytoplasmic tail region, so that the level of secretion of the S protein to the outside of a cell membrane can be increased, and a high-titer neutralizing antibody aiming at a pseudovirus is triggered.
(3) The preparation method is simple, and the required equipment is conventional equipment.
Drawings
FIG. 1 structures of different cationic polymers;
FIG. 2S.dCT DNA expression vector construction;
FIG. 3 is a schematic diagram of the preparation of a DNA nano-vaccine;
FIG. 4 DLS particle size plot of DNA nano-vaccine;
FIG. 5 transmission electron micrograph of DNA nano-vaccine (scale bar 100 nm);
FIG. 6 Western blot analysis of DNA nano-vaccine in vitro expression of S protein;
FIG. 7 shows the expression level of S protein on cell membrane;
FIG. 8 shows the level of binding antibodies in the serum of a mouse immunized with the DNA nanoball;
FIG. 9 shows the experimental results of CD4+ and CD8+ T cell immunity after the DNA nano vaccine is used for immunizing mice, wherein (A-B) is the percentage content of IFN-gamma, (C-D) is the percentage content of TNF-alpha, and (E-F) is the percentage content of IL-4;
FIG. 10 is a graph showing the results of the neutralization experiment of pseudoviruses, (A) is a fluorescence graph showing the neutralization of pseudoviruses, and (B) is a graph showing the neutralization titer of pseudoviruses;
FIG. 11 shows the safety test results of the DNA nanobacteria vaccine for mice, wherein (A) is the body weight change of the mice within 14 days after the DNA nanobacteria injection, (B) is the body temperature change of the mice within 14 days after the DNA nanobacteria injection, and (C) is the hematoxylin-eosin staining pattern (scale bar is 100 microns) of the heart, liver, spleen, lung, kidney and brain of the mice at 14 days after the DNA nanobacteria injection.
Detailed Description
The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all commercially available unless otherwise specified.
Example 1: preparation of cationic polymers
In the embodiment, N-butylamine and L-aspartic acid-5-benzyl ester-N-carboxylic acid anhydride (BLA-NCA) monomers are used as raw materials to obtain high-molecular PBLA through ring-opening polymerization reaction, and amino micromolecules are introduced into a PBLA side chain through ammonolysis reaction to synthesize the cationic polymer. Wherein the amino micromolecule can be Ethylenediamine (EDA), Diethylenetriamine (DET), triethylene tetramine (TET), Tetraethylenepentamine (TEP), bis 3-aminopropyl-1, 3-propanediamine (BAP), tris (2-aminoethyl) amine (TAE) or tris (3-aminopropyl) amine (TAP), and correspondingly, the cationic polymer [ N- (2-aminoethyl) aspartic acid ] can be obtained and is abbreviated as PASp (EDA); poly { N- [ N ' - (2-aminoethyl) -2-aminoethyl ] aspartic acid } abbreviated as PASP (DET), poly (N- { N ' - [ N "- (2-aminoethyl) -2-aminoethyl ] -2-aminoethyl } aspartic acid) abbreviated as PASP (TET), poly [ N- (N ' - { N" - [ N ' "- (2-aminoethyl) -2-aminoethyl ] -2-aminoethyl } -2-aminoethyl) aspartic acid ] abbreviated as PASP (TEP), poly (N- { N ' - [ N" - (3-aminoethyl) -3-aminoethyl ] -3-aminoethyl } aspartic acid) abbreviated as PASP (BAP), poly (N, N, N-3 (2-aminoethyl) aspartic acid) abbreviated as PASP (TAE), or poly (N, N, N-3 (3-aminopropyl) aspartic acid) is abbreviated as PASP (TAP). The structures of the different cationic polymers are shown in figure 1.
Example 2: construction of dCT DNA expression vector
According to the official published SARS-CoV-2S protein sequence (Wuhan/WIV04/2019), 99 nucleotides (SEQ ID No.1) of the last cytoplasmic tail region of the sequence are deleted, EcoRI and Bg1II enzyme cutting sites are respectively added at the 5 'end and the 3' end of the cDNA fragment, and the cDNA fragment is cloned into a pCAGGS expression vector containing a chicken beta-actin promoter and under the control of a beta-globin polyadenylation signal, wherein the resistance gene is an ampicillin-resistant gene.
Example 3: preparation of DNA nano vaccine
Taking cationic polymer PASP (EDA) as an example to prepare the DNA nano-vaccine, the PASP (EDA) and the S.dCT DNA expression vector are respectively dissolved in HEPES buffer solution with the concentration of 10mM and the pH value of 7.40, so that the concentration of the cationic polymer is 1.0 to 10 mu g/mu L, and the concentration of the S.dCT DNA expression vector is 0.1 to 10 mu g/mu L.
In this example, the concentration of S.dCT DNA expression vector was 0.1. mu.g/. mu.L, and the concentration of PASp (EDA) solution was adjusted according to the N/P ratio (molar ratio of protonatable nitrogen atoms in cationic polymer to phosphate groups in S.dCT DNA expression vector was 1 to 60). Adding the solution of PASP (EDA) into the solution of S.dCT DNA expression vector with double volume, mixing by vortex for 30s, and standing for half an hour to prepare the solution of S.dCT/PASP (EDA) complex, namely DNA nano-vaccine. In the same manner, solutions of other cationic polymers, PASP (DET), PASP (TET), PASP (TEP), PASP (BAP), PASP (TAE), PASP (TAP) or their derivatives, can be used to prepare solutions of the complexes. A schematic diagram of the preparation of DNA nano-vaccine is shown in FIG. 3.
The particle size of 100. mu.L of the compound solution with the N/P ratio of 10:1 was measured by a Malvern laser particle sizer, and the particle size results of each group of DNA nano-vaccines are shown in FIG. 4, and the hydrated particle size was about 60-70 nm. After the above complex was negatively dyed, the morphology and size of the complex were observed by a transmission electron microscope, and the results are shown in fig. 5, in which the nanoparticles were round particles with uniform size.
Example 4: expression of DNA nano vaccine in vitro S protein
Human renal epithelial cells (293T cells) were seeded in 6-well plates at 37 ℃ with 5% CO2Incubate overnight in incubator with primary cell density of 5X 10 per well5And (4) cells. Cells were transfected with 3. mu.g of the DNA nano-vaccine of example 3 per well, after 48 hours the cells were scraped off with a cell scraper, the cells were lysed with cell lysis buffer on ice for 30 minutes, and the lysed sample was heated with loading buffer at 95 ℃ for 10 minutes to perform sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 8% separation gel and 4% concentration gel). Then, the proteins were transferred from the gel onto a 0.45 μm polyvinylidene fluoride membrane (PVDF membrane) under 300mA electrophoresis conditions, and the membrane was blocked in 5% (W/V) skim milk powder for 1 h. The PVDF membrane containing protein and anti-spike glycoprotein antibody or anti-beta-actin mouse monoclonal antibody are incubated overnight at 4 ℃, then incubated with peroxidase affinity pure goat anti-mouse IgG (H + L) secondary antibody for 1H, and finally incubated with ECL hypersensitive luminescent solution and imaged by exposure instrument. The experimental result is shown in figure 6, which shows that the DNA nano vaccine can transfect 293T cell surface in vitroAnd (3) protein S.
Example 5: flow cytometry analysis of cell membrane surface S protein expression
293T cells expressing the ACE2 receptor (293T/ACE2) were seeded in 24-well plates at an initial cell density of 5X 104One/well at 37 ℃ 5% CO2Was cultured overnight in an incubator. The DNA nano-vaccine prepared in example 3 (1. mu.g DNA/well) was added to the cells, and after 48 hours of culture, the cells were harvested and blocked with 1% BSA for 30 minutes. After blocking, the cells were washed three times with PBS and incubated with anti-S protein antibody for 1h at 4 ℃. Then, Fluorescein (FITC) was added to the cells, and goat anti-mouse IgG (H + L) was affinity-purified and incubated at 4 ℃ for 1 hour with exclusion of light. After 3 washes with PBS, cells were analyzed by flow cytometry. The experimental results are shown in fig. 7, which illustrates that the DNA nano-vaccine can secrete S protein onto cell membrane for recognition by immune cells to generate immune response.
Example 6: humoral response of mice to S protein
The DNA nano-vaccine of example 3 or Phosphate Buffered Saline (PBS) was injected intramuscularly to the legs of mice, and each mouse was injected with 25. mu.g of DNA calculated as DNA in the vaccine. The mice were immunized once every two weeks for a total of three times, and 2 weeks after the last immunization, and the bound antibody content was measured by enzyme-linked immunosorbent assay (ELISA). The 96-well plate was precoated with 1. mu.g/mL SARS-CoV-2S protein, incubated overnight at 2-8 ℃ and blocked with 2% bovine serum albumin solution for 1 hour at room temperature. Then adding heat inactivated serum with different dilution times into each well, incubating for 2 hours at 37 ℃, then adding 3, 3 ', 5, 5' -tetramethyl benzidine into each well after incubating goat anti-mouse antibody combined with horseradish peroxidase for 1 hour at 37 ℃ in the dark, and then adding 2mol/L sulfuric acid to stop the reaction. The absorbance values were read at 450nm with a microplate reader. The ELISA endpoint titer was defined as the highest dilution of serum that produced absorbance. The experimental results are shown in fig. 8, and the DNA nano-vaccine stimulates mice to produce high levels of knots and antibodies.
Example 7: DNA nano vaccine immunity induction mouse T cell immune response
Mice were immunized three times according to the immunization protocol in example 6, and two weeks after the last immunization, mice were sacrificedSpleen tissue was homogenized from mice, resuspended in PBS, and then passed through a 70 μm nylon cell filter using a mixture of overlapping peptides of SARS-CoV-2S protein (2 μ g/mL) and lymphocyte stimulating agent at 37 ℃ in 5% CO2Splenocytes were stimulated for 6 h. After incubation, cells were stained with fluorescently labeled anti-CD 3, CD4, and CD8 antibodies. After cell surface staining, cells were fixed, stained for intracellular tumor necrosis factor-alpha (TNF- α), interferon- γ (IFN- γ), and interleukin-4 (IL-4), detected using a flow cytometer, and then analyzed using FlowJo software. The results are shown in FIG. 8, and the two DNA nano-vaccines cause T cell immune response, the contents of cytokines TNF-alpha and IFN-gamma are obviously increased, and the content of IL-4 is almost unchanged, which indicates that the vaccine causes Th1 type immune response without causing the risk of antibody dependence enhancement.
Example 8: study on neutralization of SARS-CoV-2 pseudovirus by mouse immune serum
293T cells were transfected with plasmid encoding codon optimized SARS-CoV-2S protein (S.dCT DNA expression vector), PCDH-Luc-mCherry vector and psPAX2 with gag/pol expression plasmid. After 48 hours of infection, pseudoviruses expressing luciferase and red fluorescent protein (mCherry) were collected from the culture supernatant, concentrated in an ultrafiltration centrifuge tube, and stored at-80 ℃ until use.
293T/ACE2 cells were seeded at a density of 10 per well in 96-well plates4Individual cells, 5% CO at 37 ℃2Incubate overnight in the incubator. Serum samples from mice immunized in example 6 were serially diluted in 96-well plates. The same volume of pseudovirus was added to the wells, preincubated at 37 ℃ for 1h, and then added to 293T/ACE2 cells to infect the cells for 48 h. The expression of mCherry red fluorescence was observed with a fluorescent light microscope. After the cells were lysed, the luciferase content of the cells was detected using a microplate reader. The SARS-CoV-2 neutralization titer was defined as the dilution of the sample that decreased 50% relative to luciferase units (RLU) compared to the control group. The experimental result is shown in figure 10, the neutralizing antibody titer of the S.dCT/DET nano vaccine caused by mice is 1: 115, and the neutralizing antibody titer of the S.dCT/TEP nano vaccine caused by mice is 1: 143.
Example 9: safety experiment of DNA nano vaccine
Mice were immunized with DNA nano-vaccine or PBS, and each mouse was injected intramuscularly with 25 μ g of DNA, calculated as DNA, and the body temperature and body weight changes of the mice were recorded within 14 days after drug administration. Mice were sacrificed after 14 days, and their hearts, livers, spleens, lungs, kidneys and brains were stained with hematoxylin-eosin and the toxicity of the vaccine to the mouse tissues was observed. The experimental result is shown in figure 11, and the result shows that the DNA nano vaccine can not cause the change of the body temperature, the body weight and the main organ tissues of the mouse and has safety.
Sequence listing
<110> Sichuan university
<120> novel coronavirus pneumonia DNA nano vaccine and preparation method thereof
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aatgtgacct ggttccacgc catccacgtg tccggcacaa acggcacaaa gaggtttgac 240
aatcccgtgc tgcctttcaa tgacggcgtg tacttcgcca gcaccgagaa gtccaatatc 300
atcaggggct ggatcttcgg caccaccctg gatagcaaga cccagtccct gctgatcgtg 360
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ggcaagcagg gcaactttaa gaacctgagg gagtttgtgt ttaagaatat cgacggctac 600
ttcaagatct actccaagca cacacccatc aacctggtga gagacctgcc ccagggcttt 660
agcgccctgg agcccctggt ggacctgcca atcggcatca acatcaccag atttcagaca 720
ctgctggccc tgcacaggtc ctacctgaca cctggcgatt ccagctccgg ctggaccgcc 780
ggagccgctg cttactacgt gggctacctg caacccagaa cattcctgct gaagtacaac 840
gagaatggca ccatcaccga tgccgtggac tgtgccctgg accctctgtc cgagaccaag 900
tgtacactga agagctttac cgtggagaag ggcatctacc agaccagcaa cttcagggtg 960
cagcctacag agtccatcgt gaggtttcct aatatcacca atctgtgccc tttcggcgag 1020
gtgtttaacg ccacaaggtt tgcctccgtg tacgcctgga ataggaagag aatcagcaat 1080
tgtgtggccg actacagcgt gctgtacaac agcgccagct tcagcacatt caagtgttac 1140
ggcgtgtccc ccaccaagct gaacgacctg tgcttcacca acgtgtacgc cgactccttc 1200
gtgatcagag gcgatgaggt gaggcagatc gcccccggcc agacaggcaa gatcgccgac 1260
tacaactaca agctgcccga cgattttaca ggctgcgtga tcgcctggaa cagcaacaac 1320
ctggatagca aagtgggcgg caactacaac tacctgtaca ggctgttcag aaagagcaat 1380
ctgaagccct tcgagagaga tatcagcacc gagatctacc aggccggcag cacaccctgt 1440
aacggcgtgg agggctttaa ttgttacttc cccctgcaat cctacggctt ccagcccacc 1500
aatggcgtgg gctaccagcc ttacagagtg gtggtgctgt ccttcgagct gctgcacgcc 1560
cccgccaccg tgtgtggacc taagaagagc accaacctgg tgaagaacaa gtgcgtgaac 1620
tttaatttca atggcctgac cggcaccggc gtgctgacag agtccaacaa gaagtttctg 1680
cctttccagc agtttggcag agatatcgcc gatacaacag acgccgtgag agatcctcag 1740
acactggaga tcctggatat cacaccctgc tccttcggcg gcgtgtccgt gatcacacct 1800
ggcacaaata caagcaatca ggtggccgtg ctgtaccagg acgtgaattg caccgaggtg 1860
cctgtggcca tccacgccga tcagctgaca cccacatgga gagtgtacag caccggcagc 1920
aacgtgttcc agaccagagc cggctgtctg atcggcgccg agcacgtgaa taactcctac 1980
gagtgtgaca tccccatcgg cgccggcatc tgcgccagct accagacaca gacaaactcc 2040
cccaggaggg ccagatccgt ggcctcccag tccatcatcg cctacacaat gtccctgggc 2100
gccgagaact ccgtggccta ctccaacaac tccatcgcca tccctacaaa cttcacaatc 2160
agcgtgacaa cagagatcct gcccgtgtcc atgaccaaga ccagcgtgga ctgtaccatg 2220
tacatctgcg gcgatagcac cgagtgctcc aatctgctgc tgcaatacgg ctccttctgt 2280
acccagctga atagggccct gacaggcatc gccgtggagc aggacaagaa cacccaggag 2340
gtgttcgccc aggtgaagca gatctacaag acacccccta tcaaggactt cggcggcttt 2400
aactttagcc agatcctgcc tgacccttcc aagccctcca agagatcctt catcgaggat 2460
ctgctgttta ataaggtgac cctggccgat gccggcttca tcaagcagta cggcgactgc 2520
ctgggcgata tcgccgccag agacctgatc tgcgcccaga agtttaacgg cctgaccgtg 2580
ctgcctcccc tgctgaccga tgagatgatc gcccagtaca catccgccct gctggccggc 2640
acaatcacat ccggctggac attcggcgcc ggcgccgctc tgcaaatccc cttcgccatg 2700
cagatggcct acaggtttaa cggcatcggc gtgacacaga acgtgctgta cgagaatcag 2760
aagctgatcg ccaaccagtt caattccgcc atcggcaaga tccaggactc cctgtccagc 2820
accgcctccg ccctgggaaa gctgcaagac gtcgtgaatc agaacgcaca ggccctgaat 2880
actctggtga agcagctgtc ctctaacttc ggcgccatta gttcagtgct gaatgatatc 2940
ctgagccggc tggacaaagt cgaggctgaa gtgcagattg accgcctgat cacagggcga 3000
ctgcagagcc tgcagactta tgtgacccag cagctgattc gggctgcaga aatcagagct 3060
agcgcaaatc tggccgctac caagatgtct gagtgcgtcc tgggccagag taagagagtg 3120
gacttttgtg ggaaaggata tcacctgatg tcattcccac agagcgcccc tcacggagtc 3180
gtgtttctgc atgtcaccta cgtgccagct caggagaaga acttcactac cgcccccgct 3240
atctgccacg atggcaaagc ccattttcct agggaaggcg tcttcgtgtc caacgggact 3300
cattggtttg tgacccagcg caatttctac gagccacaga tcattacaac tgacaatacc 3360
ttcgtgtctg gaaactgtga tgtcgtgatt ggcatcgtca acaatacagt gtatgatcct 3420
ctgcagccag agctggactc ctttaaggag gaactggata agtacttcaa aaatcacacc 3480
tctcccgacg tggatctggg ggacatttct ggaatcaatg caagtgtcgt gaacattcag 3540
aaggagatcg acaggctgaa cgaagtggcc aaaaatctga acgagtccct gatcgatctg 3600
caggagctgg gcaagtatga acagtacatc aagtggccct ggtacatttg gctgggcttc 3660
atcgcagggc tgattgccat cgtcatggtg accatcatgc tgtgctgtat gacatcttgc 3720
tgtagttgcc tgaaggggtg ctgttcatgt ggaagctgct gttaa 3765