CN113521272B - Novel coronavirus pneumonia DNA nano vaccine and preparation method thereof - Google Patents

Abstract

The invention discloses a DNA nano vaccine for novel coronavirus pneumonia and a preparation method thereof, wherein a DNA cytoplasmic tail region of spike protein (S) on a traditional encoding novel coronavirus shell is deleted to be S.dCT DNA, and the DNA cytoplasmic tail region is self-assembled with a cationic polymer through electrostatic interaction to form the nano vaccine, wherein the cationic polymer is one of PAsp (EDA), PAsp (DET), PAsp (TET), PAsp (TEP), PAsp (BAP), PAsp (TAE) or PAsp (TAP) or derivatives of the same type. 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 trigger high-titer neutralizing antibodies against pseudoviruses, and has high safety, thus being applicable to preventing and controlling novel coronavirus pneumonia.

Description

Novel coronavirus pneumonia DNA nano vaccine and preparation method thereof

Technical Field

The invention belongs to the field of vaccines, and in particular relates to a novel coronavirus pneumonia DNA nanometer vaccine and a preparation method thereof.

Background

The novel coronavirus (Severe Acute Respiratory Syndrome Coronavirus, SARS-CoV-2) is the cause of outbreaks of the novel coronavirus pneumonia (COVID-19), and epidemic situation continuously outbreaks and persists worldwide from 2019, resulting in huge casualties and great economic losses. In the face of recurrent epidemic and infection, only vaccines are the most effective weapons against viruses (Vaccine 39 (2) (2021) -197-201).

The protein coat of SARS-CoV-2 consists of spike protein (S), membrane glycoprotein (M), nucleocapsid protein (N), hemagglutinin esterase dimer egg (He) and envelope protein (E). The S protein is a virus fusion protein, mediates the adhesion of viruses to angiotensin converting enzyme 2 (ACE 2) receptors on the cell surface, performs membrane fusion, and is absorbed by endosomes after entering cells. Proteolytic cleavage of the S protein and fusion of the viral membrane with the endosomal membrane triggers release of viral RNA into the cytoplasm (Nat Microbiol 5 (4) (2020) 562-569). 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 critical target for developing SARS-CoV-2 vaccine.

Currently, various SARS-CoV-2 candidate vaccines are being developed, which are mainly divided into five technical directions: inactivated vaccine, recombinant protein vaccine, adenovirus vector vaccine, attenuated influenza virus vector vaccine and nucleic acid vaccine (which are 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 the company of Buddha, U.S. and Germany, and mRNA-1273 et al (N.Engl. J. Med.383 (20) (2020) 1920-1931) developed by the company of Morgana, U.S. however, existing vaccines have a short protection period, large side reaction and safety problems, and in addition, the continuous variation of viruses requires the development of novel crown vaccines.

The DNA vaccine technology is relatively mature, plasmid DNA can exist in a host for a long time, antigen genes are continuously expressed in the host to generate antigen proteins, the immune system of the organism is continuously stimulated to generate long-range immunity, the immune effect is reliable, the gene vaccine can generate humoral immune response, and can cause activation of cytotoxic T lymphocytes to induce cellular immunity, and the traditional vaccine only can induce cellular immunity by a live vaccine, but has the danger of virulence rebound of the live vaccine. DNA vaccines are safer than inactivated vaccines that require in vitro culture of coronaviruses, and the inactivated vaccine is at risk of eliciting an enhanced antibody-dependent infection (ADE-dependent enhancement of infection, ADE), the ability of antibodies to increase viral infection, ultimately exacerbating the disease (Nature Microbiology 5 (10) (2020) 1185-1191). DNA is more stable, temperature stable and not cold chain limited than mRNA vaccines, an important advantage for delivery into environments where resources are limited.

Gene delivery vectors are a critical part of DNA vaccines, and mainly include two major classes, viral and non-viral vectors. Although the transfection efficiency of viral vectors is high, the development and application of viral vectors are greatly limited due to the difficulty in preparation and production, high immunogenicity, limited size of the entrapped genes, and the like. Non-viral vectors primarily include cationic polymers and cationic liposomes that deliver genes into cells through electrostatic interactions with the genes. The cationic polymer is easy to synthesize and modify and has no immunogenicity, so that the cationic polymer can be used as a good gene delivery carrier for preparing novel coronavirus pneumonia DNA nano vaccine, but the transfection efficiency, stability and safety of the novel coronavirus pneumonia DNA nano vaccine are concerned, and particularly the problems of ADE effect generation and the like are avoided.

Walls et al (Cell 183 (5) (2020) 1367) constructed plasmids expressing the S protein receptor binding domain, and prepared S protein nanoparticle vaccines by transfecting 293 cells with plasmids, extracting and purifying the S protein receptor binding domain from the 293 cells, and inducing potent neutralizing antibody responses in BALB/c mice. This technique has the following disadvantages:

(1) The purification process of extracting and purifying protein and preparing nano particles is more complicated, and the production cost is higher;

(2) There is no concern whether the vaccine is safe or not, and if it will cause ADE effects.

Smith et al (Nature Communications 11 (1) (2020)) constructed a plasmid expressing S protein, extracted the DNA of interest, injected into the mouse tibial anterior muscle, and then pulsed with constant current using in vivo electroporation equipment to facilitate transfection of DNA into cells of mice, which DNA vaccine stimulated the mice to produce neutralizing antibodies, blocking binding of S protein to ACE2 receptor, and preventing infection by 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, most of which is retained on the endoplasmic reticulum membrane (Journal of Virology,2020.94 (21)), and less is secreted outside the cell;

(2) The special electroporation equipment is needed to promote the transfection of DNA, so that the cost is high;

(3) Conditions for electroporation are controlled, otherwise cell death rate is high or transfection efficiency is low.

Disclosure of Invention

The technical problems to be solved by the invention are as follows:

(1) Aiming at the current situation of the novel coronavirus epidemic situation, a vaccine for preventing the novel coronavirus infection 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 can secrete the S protein to the outside of cells for being identified by immune cells so as to cause strong immune response is constructed.

(2) Provides a preparation method of a novel coronavirus pneumonia DNA nanometer vaccine.

(3) Provides the application of the novel coronavirus pneumonia DNA nanometer vaccine.

The technical scheme of the invention is as follows: a novel coronavirus pneumonia DNA nano vaccine is formed by electrostatic self-assembly of a plasmid vector and a cationic polymer, wherein the plasmid vector contains DNA which codes novel coronavirus shell S protein and deletes 99 nucleotides in cytoplasmic tail region, and the cationic polymer is polyaspartic acid.

Further, the nucleotide sequence of DNA encoding novel coronavirus coat S protein and deleted of cytoplasmic tail region of 99 nucleotides is shown in SEQ ID No. 1.

Further, the plasmid vector is a pCAGGS vector.

Further, the molar ratio of the protonatable nitrogen atoms in the cationic polymer to the phosphate groups in the plasmid vector is from 1 to 60.

Further, the cationic polymer poly [ N- (2-aminoethyl) aspartic acid ] is abbreviated as PAsp (EDA) and has a molecular weight of 1.0-40kDa; poly { N- [ N' - (2-aminoethyl) -2-aminoethyl ] aspartic acid } abbreviated as PAsp (DET) and has a molecular weight of 1.0-40kDa; poly (N- { N '- [ N' - (2-aminoethyl) -2-aminoethyl ] -2-aminoethyl } aspartic acid) abbreviated as PAsp (TET) and has a molecular weight of 1.0 to 40kDa; poly [ N- (N '- { N "- [ N'" - (2-aminoethyl) -2-aminoethyl ] -2-aminoethyl } -2-aminoethyl) aspartic acid ] is abbreviated as PAsp (TEP) and has a molecular weight of 1.0 to 40KDa; poly (N- { N '- [ N' - (3-aminoethyl) -3-aminoethyl ] -3-aminoethyl } aspartic acid) abbreviated as PAsp (BAP) and has a molecular weight of 1.0-40kDa; poly (N, N, N-3 (2-aminoethyl) aspartic acid) is abbreviated as PAsp (TAE) and has a molecular weight of 1.0-40kDa; or poly (N, N, N-3 (3-aminopropyl) aspartic acid) abbreviated as PAsp (TAP) with a molecular weight of 1.0-40kDa.

The preparation method of the novel coronavirus pneumonia DNA nano vaccine comprises the following steps:

(1) The plasmid vector and the cationic polymer were each formulated into a solution of appropriate concentration with hydroxyethylpiperazine ethylsulfuric acid (HEPES) buffer solution at pH 7.40.

(2) Uniformly mixing the plasmid carrier solution and the cationic polymer solution according to a proper proportion, standing, and enabling the positively charged cationic polymer and the negatively charged DNA to form the DNA nano vaccine through electrostatic interaction, wherein the particle size of the DNA nano vaccine is 40-100nm.

Further, the concentration of the cationic polymer is 1.0-10. Mu.g/. Mu.L, and the concentration of the plasmid vector is 0.1-10. Mu.g/. Mu.L; the molar ratio of protonatable nitrogen atoms in the cationic polymer to phosphate groups in the plasmid vector (N/P ratio) is 1-60.

The DNA nanometer vaccine of the invention is applied to the preparation of medicines for preventing or/and treating 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 to remove a cytoplasmic tail region, so that the secretion level of the S protein outside a cell membrane can be increased, and a neutralizing antibody with high titer aiming at a pseudovirus is initiated.

(3) The preparation method is simple, and the required equipment is conventional equipment.

Drawings

FIG. 1 shows the structure of different cationic polymers;

construction of a dCT DNA expression vector;

FIG. 3 is a schematic diagram of the preparation of DNA nanovaccines;

FIG. 4 DLS particle size diagram of DNA nanovaccine;

FIG. 5 transmission electron microscopy of DNA nanovaccine (scale bar 100 nm);

FIG. 6 Western blot analysis of in vitro expression of S protein by DNA nanovaccine;

FIG. 7 amount of expression of protein S on cell membrane;

FIG. 8 content of binding antibodies in mouse serum after immunization of mice with DNA nanovaccine;

FIG. 9 experimental results of CD4+ and CD8+ T cell immunity after immunization of mice with DNA nanovaccine, wherein (A-B) is the percentage of IFN-gamma, (C-D) is the percentage of TNF-alpha, and (E-F) is the percentage of IL-4;

FIG. 10 shows the results of pseudo-virus neutralization assay, (A) shows pseudo-virus neutralization fluorescence, and (B) shows pseudo-virus neutralization titer;

FIG. 11 safety test results of DNA nanovaccine on mice, wherein (A) is a graph of body weight change of mice within 14 days after injection of DNA nanovaccine, (B) is a graph of body temperature change of mice within 14 days after injection of DNA nanovaccine, and (C) is a graph of hematoxylin-eosin staining of heart, liver, spleen, lung, kidney and brain of mice on 14 days after injection of DNA nanovaccine (scale bar 100 μm).

Detailed Description

The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from commercial sources.

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 small molecules are introduced into PBLA side chains through ammonolysis reaction to synthesize the cationic polymer. Wherein the small amino molecule can be Ethylenediamine (EDA), diethylenetriamine (DET), triethylenetetramine (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 ] abbreviated PAsp (EDA) can be obtained; poly { N- [ N '- (2-aminoethyl) -2-aminoethyl ] aspartic acid } is abbreviated as PAsp (DET), poly (N- { N' - [ N "- (2-aminoethyl) -2-aminoethyl ] -2-aminoethyl } aspartic acid) is abbreviated as PAsp (TET), poly [ N- (N '- { N" - [ N "- (2-aminoethyl) -2-aminoethyl ] -2-aminoethyl } -2-aminoethyl) aspartic acid ] is abbreviated as PAsp (TEP), poly (N- { N' - [ N" - (3-aminoethyl) -3-aminoethyl ] -3-aminoethyl } aspartic acid) is abbreviated as PAsp (BAP), poly (N, N-3 (2-aminoethyl) aspartic acid) is abbreviated as PAsp (TAE), or poly (N, N-3 (3-aminopropyl) aspartic acid) is abbreviated as PAsp (TAP). The structure of the different cationic polymers is shown in figure 1.

Example 2: construction of S.dCT DNA expression vector

According to the officially published SARS-CoV-2S protein sequence (Wuhan/WIV 04/2019), the last cytoplasmic tail region of the sequence was deleted 99 nucleotides (SEQ ID No. 1), ecoRI and Bg1II cleavage sites were added to the 5 'and 3' ends of its cDNA fragment, respectively, and cloned into a pCAGGS expression vector under the control of chicken beta-actin promoter and beta-globin polyadenylation signal, wherein the resistance gene is an ampicillin resistance gene.

Example 3: preparation of DNA nanovaccine

Taking cationic polymer PAsp (EDA) as an example to prepare a DNA nano vaccine, respectively dissolving PAsp (EDA) and S.dCT DNA expression vectors in 10mM HEPES buffer solution with pH of 7.40 to ensure that the concentration of the cationic polymer is 1.0-10 mug/mug and the concentration of the S.dCT DNA expression vector is 0.1-10 mug/mug.

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 different N/P ratios (molar ratio of protonatable nitrogen atoms in the cationic polymer to phosphate groups in the S.dCT DNA expression vector was 1-60). The PAsp (EDA) solution was added to twice the volume of the S.dCT DNA expression vector solution, vortexed and mixed for 30s, and left to stand for half an hour to prepare the S.dCT/PAsp (EDA) complex solution, i.e., DNA nanovaccine. Using the same procedure, complex solutions can be prepared using other cationic polymers PAsp (DET), PAsp (TET), PAsp (TEP), PAsp (BAP), PAsp (TAE), PAsp (TAP), or derivatives thereof. A schematic diagram of the preparation of DNA nanovaccine 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 Markov laser particle sizer, and the particle size results of each group of DNA nanovaccines are shown in FIG. 4, and the hydrated particle sizes are all about 60-70 nm. After the above-mentioned complex is negatively stained, the morphology and size thereof are observed by a transmission electron microscope, and as a result, the nanoparticles are round particles having a uniform size, as shown in fig. 5.

Example 4: expression of S protein in vitro of DNA nano vaccine

Human kidney epithelial cells (293T cells) were seeded in 6-well plates at 37℃with 5% CO ₂ Incubation overnight in incubator with a primary cell density of 5×10 cells per well ⁵ Individual cells. Cells were transfected with a DNA nanovaccine containing 3 μg of example 3 per well, scraped off with a cell scraper after 48h, lysed with cell lysate on ice for 30 min, and the lysed samples were heated with loading buffer at 95 ℃ for 10 min for sulfate-polyacrylamide gel electrophoresis of sodium dodecyl (SDS-PAGE, 8% separation gel and 4% concentration gel). The proteins were then 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) skimmed milk powder for 1h. The PVDF membrane containing the protein is incubated with an anti-spike glycoprotein antibody or a mouse monoclonal antibody against beta-actin at 4 ℃ overnight, then with a peroxidase affinity pure goat anti-mouse IgG (H+L) secondary antibody for 1H, and finally, the ECL hypersensitive luminescence liquid is used for incubation and then an exposure instrument is used for imaging. The experimental result is shown in fig. 6, which shows that the DNA nano vaccine can express S protein by transfecting 293T cells in vitro.

Example 5: flow cytometry analysis of cell membrane surface S protein expression

293T cells expressing ACE2 receptor (293T/ACE 2) were seeded in 24-well plates at an initial cell density of 5X 10 ⁴ Individual wells at 37 ℃,5% co ₂ Is cultured overnight in an incubator. The DNA nanovaccine (1. Mu.g DNA/well) prepared in example 3 was added to the cells, and after culturing for 48 hours, the cells were harvested and blocked with 1% BSA for 30 minutes. After blocking, cells were washed three times with PBS and incubated with anti-S protein antibody for 1h at 4 ℃. Then, fluorescein (FITC) coupled affinity purified goat anti-mouse IgG (H+L) was added and incubated at 4℃for 1H in the absence of light. After 3 washes with PBS, the cells were analyzed with a flow cytometer. The experimental results are shown in fig. 7, which illustrates that the DNA nanovaccine 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 nanovaccine or Phosphate Buffered Saline (PBS) in example 3 was injected intramuscularly in the legs of mice, each mouse injected with 25 μg of DNA, calculated as DNA in the vaccine. The mice were immunized once every two weeks, three times in total, and the serum of the mice was taken 2 weeks after the last immunization, and the content of the bound antibodies was tested by enzyme-linked immunosorbent assay (ELISA). The 96-well plates were precoated with 1. Mu.g/mL SARS-CoV-2S protein, incubated overnight at 2-8deg.C, and blocked with 2% bovine serum albumin solution for 1 hour at room temperature. Then, heat-inactivated serum with different dilution factors is added to each well to incubate for 2 hours at 37 ℃, then goat anti-mouse antibody combined with horseradish peroxidase is incubated for 1 hour at 37 ℃ in dark place, 3', 5' -tetramethylbenzidine is added to each well, and then 2mol/L sulfuric acid is added to stop the reaction. Absorbance values were read with a microplate reader at 450 nm. ELISA endpoint titer was defined as the highest serum dilution giving absorbance. The experimental results are shown in fig. 8, and the DNA nanovaccine stimulated mice to produce high levels of binding and antibodies.

Example 7: immunization of DNA nanovaccine induces T cell immune response in mice

After three times immunization of mice according to the immunization protocol in example 6, two weeks after the last immunization, mice were sacrificed to remove spleen, spleen tissue was homogenized, resuspended in PBS, and then passed through a 70 μm nylon cell filter with overlapping peptides of SARS-CoV-2S protein (2. Mu.g/mL) and lymphocyte stimulating agent at 37℃in 5% CO ₂ Spleen cells were stimulated for 6h. After incubation, cells were stained with fluorescently labeled anti-CD 3, CD4 and CD8 antibodies. After staining the cell surface, the cells were fixed, and after staining for intracellular tumor necrosis factor- α (TNF- α), interferon- γ (IFN- γ) and interleukin-4 (IL-4), they were detected using a flow cytometer and then analyzed using FlowJo software. The results are shown in FIG. 8, where the two DNA nanovaccines elicit T cell immune responses with significantly increased levels of cytokines TNF- α and IFN- γ, while the levels of IL-4 are almost unchanged, indicating that the vaccine elicits Th1 type immune responses without risking increased antibody dependence.

Example 8: study of mice immune serum neutralization of SARS-CoV-2 pseudovirus

293T cells were transfected with SARS-CoV-2S protein plasmid (S.dCT DNA expression vector), PCDH-Luc-mCherry vector and psPAX2 with gag/pol expression plasmid encoding the codons. After 48 hours of infection, pseudoviruses expressing luciferase and red fluorescent protein (mCherry) were collected from the culture supernatant, concentrated in ultrafiltration tubes and stored at-80 ℃ until use.

293T/ACE2 cells were seeded in 96-well plates at a density of 10 per well ⁴ Individual cells, at 37 ℃,5% co ₂ Incubate overnight in incubator. Serum samples of mice immunized in example 6 were serially diluted in 96-well plates. The same volume of pseudovirus was added to the wells, pre-incubated for 1h at 37℃and then added to 293T/ACE2 cells to infect the cells for 48h. The expression of mCherry red fluorescence was observed with a fluorescence light microscope. After lysing the cells, the luciferase content of the cells was measured using an enzyme-labeled instrument. SARS-CoV-2 neutralization titer is defined as the 50% reduction in sample dilution relative to luciferase units (RLU) over the control. The experimental results are shown in FIG. 10, wherein the titer of the neutralizing antibody generated by the S.dCT/DET nano vaccine is 1:115, and the titer of the neutralizing antibody generated by the S.dCT/TEP nano vaccine is 1:143.

Example 9: DNA nanovaccine safety experiment

Mice were immunized with either DNA nanovaccine or PBS, each mouse was intramuscular injected with 25 μg of DNA, and body temperature and body weight changes of the mice were recorded within 14 days after dosing. Mice were sacrificed after 14 days and their hearts, livers, spleens, lungs, kidneys and brains were hematoxylin-eosin stained for toxicity of the vaccine to the mouse tissues. 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 mice, and has safety.

Sequence listing

<110> university of Sichuan

<120> novel coronavirus pneumonia DNA nanovaccine and method for preparing the same

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3765

<212> DNA

<213> novel coronavirus S protein (Artificial Sequence)

<400> 1

atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc agtgcgtgaa tctgaccaca 60

agaacccagc tgcctcccgc ctacacaaac agcttcacaa gaggcgtgta ctaccccgac 120

aaggtgttca ggagcagcgt gctgcactcc acccaggatc tgttcctgcc ctttttctcc 180

aatgtgacct ggttccacgc catccacgtg tccggcacaa acggcacaaa gaggtttgac 240

aatcccgtgc tgcctttcaa tgacggcgtg tacttcgcca gcaccgagaa gtccaatatc 300

atcaggggct ggatcttcgg caccaccctg gatagcaaga cccagtccct gctgatcgtg 360

aataacgcca ccaatgtggt cattaaggtg tgtgagttcc agttttgtaa tgaccctttt 420

ctgggcgtgt actatcacaa gaacaataag agctggatgg agtccgagtt tagggtgtac 480

agcagcgcca acaactgtac ctttgagtac gtgtcccagc ctttcctgat ggacctggag 540

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

Claims (6)

1. A novel coronavirus pneumonia DNA nanometer vaccine is formed by static self-assembly of a plasmid vector and a cationic polymer, wherein the plasmid vector contains DNA which codes novel coronavirus shell S protein and deletes 99 nucleotides in cytoplasmic tail region, the nucleotide sequence of the DNA is shown as SEQ ID No.1, the cationic polymer is poly { N- [ N ' - (2-aminoethyl) -2-aminoethyl ] aspartic acid } or poly [ N- (N ' - { N ' ' - [ N ' ' ' - (2-aminoethyl) -2-aminoethyl ] -2-aminoethyl } -2-aminoethyl) aspartic acid ] with molecular weight of 1-40K.

2. The DNA nanovaccine of claim 1, wherein the plasmid vector is a pCAGGS vector.

3. The DNA nanovaccine of claim 1, wherein the molar ratio of protonatable nitrogen atoms in the cationic polymer to phosphate groups in the plasmid vector is 1-60.

4. A method for preparing the novel coronavirus pneumonitis DNA nanovaccine of any one of claims 1-3, comprising the steps of:

(1) Preparing a plasmid vector and a cationic polymer into a solution with proper concentration by using a hydroxyethyl piperazine ethylsulfuric acid buffer solution with the pH of 7.40 respectively;

(2) Uniformly mixing the plasmid carrier solution and the cationic polymer solution according to a proper proportion, and standing to enable the positively charged cationic polymer and the negatively charged plasmid carrier to form the DNA nanometer vaccine through electrostatic interaction.

5. The method for preparing a novel coronavirus pneumo DNA nanovaccine as claimed in claim 4, wherein the concentration of the cationic polymer in the step (1) is 1.0-10. Mu.g/. Mu.L, and the concentration of the plasmid vector is 0.1-10. Mu.g/. Mu.L.

6. Use of the DNA nanovaccine of any of claims 1-3 in the preparation of a medicament for the prevention or/and treatment of novel coronavirus pneumonia.

Images