Coding gene of Zaire Ebola virus nucleocapsid protein, related mRNA molecule and application thereof

Abstract

The invention belongs to the technical field of nucleic acid vaccines, and particularly relates to a coding gene of a Zaire Ebola virus nucleocapsid protein, a related mRNA molecule and application thereof. The nucleotide sequence of the gene is as shown in SEQ ID NO.1. The DNA vaccine is prepared by using a vector of the gene. A transcription template DNA of the mRNA molecule is formed by sequentially connecting a 5 'UTR region, a gene as shown in SEQ ID NO.1, a 3' UTR region and a PolyA tail, all uridine in the mRNA molecule is replaced by N1-methyl-pseudouridine-5 '-triphosphate, and the mRNA molecule is encapsulated in lipid nanoparticles to form the mRNA vaccine; immunological evaluation is carried out by using the vaccine to immunize animals, it is proved that candidate nucleic acid vaccines can induce immune mouse antigen-specific body fluid and cellular immune response, and the potential of the novel EBOV nucleic acid vaccine in prevention of Ebola virus diseases is proved.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid vaccines, and in particular relates to a Zaire Ebola virus nucleocapsid protein encoding gene, related mRNA molecules and uses thereof. Background Art

[0002] In 1976, the Ebola virus was first discovered near the Ebola River in Zaire (now the Democratic Republic of the Congo) in Africa and was named after the river. The virus is a deadly pathogen that causes Ebola virus disease (EVD), which has severe symptoms and a high mortality rate, and has attracted widespread attention and research in the field of public health. Since the virus was first discovered, more than 20 Ebola outbreaks have occurred in sub-Saharan Africa, mainly concentrated in Sudan, Uganda, the Democratic Republic of the Congo and Gabon. Ebola virus has great epidemic potential. Zaire Ebola virus (EBOV), Sudan Ebola virus and Bundibugyo Ebola virus are the three main strains that have caused large-scale outbreaks of Ebola disease in Africa. Among them, EBOV is considered the most deadly and is the strain that caused the large-scale Ebola hemorrhagic fever outbreaks in West Africa from 2013 to 2016. The Ebola outbreak in West Africa from 2013 to 2016 was unprecedented in scale, with more than 28,000 confirmed cases and 11,000 deaths. In previous outbreaks, case fatality rates have ranged from 25% to 90%, depending on the severity of the outbreak and the effectiveness of response measures. Ebola virus disease is considered a zoonosis, occasionally spreading to humans, apes, and possibly other animals. EVD is mainly transmitted through close contact with infected patients, including contact with infected patients' body fluids such as blood, saliva, sweat, urine, or semen. There is currently no evidence of droplet infection.

[0003] Ebola virus is a single-stranded negative-sense RNA virus belonging to the Filoviridae family and the genus Filovirus. Its genome is about 18.9 kb long and contains the coding sequences of 7 structural proteins and one non-structural protein, which are the 3' non-coding region, nucleocapsid protein (NP), viral particle protein 35 (VP35), VP40, glycoproteins (sGP and GP), VP30, VP24, RNA-dependent RNA polymerase (L protein) and 5' non-coding region. The virus structure is a long, filamentous particle with a diameter of about 80 nanometers and a length of up to several microns. It is well known that activated T cells, especially CD8 + T cells play an important role in protective immunity against EBOV. Existing studies have confirmed that NP is CD8 + The preferred target of T cell attack, and NP-specific T cell response dominated the immune response. Vaccines designed against EBOV NP can rapidly induce CD8 +T cells produce NP-specific IFN-γ. Therefore, NP is considered to be a more suitable target for CTL vaccines to better simulate the immune response of infected people and enhance cellular immunity in vaccination. In addition, NP plays a key role in Ebola virus replication, making it an essential component of the viral life cycle. Compared with VP35 and VP24, NP is more likely to remain highly conserved and is therefore more likely to become an ideal target for CTL vaccines. However, the translation quality and safety of the natural NP encoding gene need to be further improved. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a gene encoding EBOV NP, and two candidate nucleic acid vaccines pVAX-NP prepared using the gene EBOV (NP D ) and EBOV NP mRNA (NP R ). The immunogenicity of the EBOV candidate nucleic acid vaccine was evaluated in BALB / c mice by intramuscular injection of each candidate vaccine. Each group of EBOV NP candidate vaccines induced antigen-specific IgG antibodies and antigen-specific Th1 cellular immune responses.

[0005] The specific technical solutions provided by the present invention are as follows:

[0006] In a first aspect, the present invention provides a gene encoding a Zaire Ebola virus nucleocapsid protein, the nucleotide sequence of the gene is shown in SEQ ID NO.1. The present invention performs human-preferred codon optimization based on the nucleotide sequence of the NP encoding gene of the EBOV Makona strain, and its codons have human-preferred characteristics compared with the NP nucleotide sequence of the EBOV Makona strain. Compared with the original gene, it has the advantages of improving protein translation quality and strong safety.

[0007] The second aspect of the present invention provides a vector containing the gene.

[0008] In a specific embodiment of the present invention, the vector is a pVAX1 vector, and the vector containing the gene is constructed by inserting the gene shown in SEQ ID NO.1 into the BamH I and Xho I sites of the pVAX1 vector.

[0009] The third aspect of the present invention provides use of the vector in preparing a DNA vaccine for preventing Ebola virus.

[0010] In a fourth aspect, the present invention provides a DNA vaccine for preventing Ebola virus, wherein the DNA vaccine contains the above-mentioned vector.

[0011] In a fifth aspect, the present invention provides an mRNA molecule expressing the nucleocapsid protein of Zaire Ebola virus, which is formed by sequentially connecting the 5′UTR region, the gene encoding the Zaire Ebola virus envelope glycoprotein, the 3′UTR region and the PolyA tail;

[0012] The mRNA molecule comprises a modified nucleoside; the modified nucleoside is a pseudouridine, the pseudouridine is N1-methyl-pseudouridine-5′-triphosphate, and all uridines in the mRNA molecule are replaced by N1-methyl-pseudouridine-5′-triphosphate.

[0013] Preferably, the 5′UTR and 3′UTR sequences are derived from Homo sapiens hemoglobin subunit β, the 5′UTR sequence includes a regulatory sequence and the 5′UTR sequence of β-globin, and the 3′UTR sequence consists of the tandem sequence of two fragments of the 3′UTR of β-globin.

[0014] Preferably, the PolyA tail is 120 nt in length.

[0015] In a sixth aspect, the present invention provides the use of the mRNA molecule expressing the Zaire Ebola virus nucleocapsid protein in the preparation of an mRNA vaccine for preventing Ebola virus.

[0016] In a seventh aspect, the present invention provides an mRNA vaccine for preventing Ebola virus, comprising the mRNA molecule expressing the Zaire Ebola virus envelope glycoprotein and at least one pharmaceutically acceptable excipient.

[0017] Preferably, the mRNA molecule is formulated into lipid nanoparticles.

[0018] In an eighth aspect, the present invention provides a method for preparing the mRNA vaccine for preventing Ebola virus, wherein the mRNA molecule expressing the nucleocapsid protein of Zaire Ebola virus, ALC-0315, DSPC, cholesterol and DMG-PEG 2000 are mixed to prepare lipid nanoparticles to obtain the mRNA vaccine.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention uses the optimized NP nucleotide sequence of the EBOV Makona strain to construct pVAX-NP expressing the molecule. EBOV The two nucleic acid vaccines, EBOV NP mRNA and EBOV NP mRNA, were then used to immunize mice, demonstrating that the constructed vaccines had anti-EBOV specific cellular and humoral immune responses and antiviral protection efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 pVAX1-NPEBOV Plasmid restriction electrophoresis verification.

[0022] Figure 2 For in vitro transcription of pGEM-UTR-NP EBOV Capillary electrophoresis of the obtained EBOV NP mRNA. (The 20nt peak represents the marker).

[0023] Figure 3 Representative intensity-size graphs of mRNA(EBOV NP)-LNP measured by dynamic light scattering.

[0024] Figure 4 TEM image of mRNA(EBOV NP)-LNP;

[0025] Figure 5 The EBOV NP-specific IgG antibody titer of the candidate nucleic acid vaccine was determined by enzyme-linked immunosorbent assay (ELISA) (n=8).

[0026] Figure 6 Specific IFN-γ SFCs were determined by ELISpot under stimulation with EBOV NP 9-peptide mixture (n=3).

[0027] Figure 7 HE staining of major organs of mice after immunization with the long-acting candidate vaccine. DETAILED DESCRIPTION

[0028] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0029] Example 1

[0030] Coding gene design and vaccine candidate construction

[0031] (1) Construction of DNA vaccine vector

[0032] Codon optimization was performed based on the NP sequence of EBOV Makona published in GenBank (MG572234.1). The target sequence was optimized for human-biased codons using the JCat tool to obtain a gene fragment as shown in SEQ ID NO.1, which encodes the antigen shown in SEQ ID NO.3. After optimization, the CAI of the NP sequence was increased from 0.19 to 0.95. Qingke Bio was commissioned to perform standard gene synthesis based on the designed sequence, which was then inserted into the Kan-resistant pVAX1 vector and transformed into the Escherichia coli TOP10 strain for delivery. After gene synthesis, the punctured bacteria containing the target plasmid were plated, single clones were picked and expanded for culture, sent for sequencing verification, bacterial strains were retained, and plasmids were purified. pVAX1-NP was verified by sequencing and electrophoresis after enzyme digestion. EBOV , the accuracy and integrity of the plasmid, and the results are as follows Figure 1 shown.

[0033] (2) Construction of mRNA vaccine vector

[0034] The gene fragment shown in SEQ ID NO.3 is formed by connecting the 5′UTR region, the gene fragment shown in SEQ ID NO.1, the 3′UTR region and the polyadenylic acid (PolyA) tail in order. The 5′UTR region and the 3′UTR region are both derived from the hemoglobin subunit β of Homo sapiens. The 5′UTR region includes a regulatory sequence and the 5′UTR sequence of β-globin. The 3′UTR region consists of a tandem sequence of two identical fragments of the 3′UTR of β-globin. The polyadenylic acid tail has a length of 120 nt.

[0035] The gene fragment shown in SEQ ID NO.3 was inserted between the BamH I and XhoI sites of the pGEM vector (Promega; P2411) to construct an in vitro expression vector - pGEM-UTR-NP EBOV Vector. pGEM-UTR-NP was obtained by in vitro transcription EBOV The vector is further capped to obtain the final mRNA molecule. The specific operation is as follows:

[0036] 1) Linearization of in vitro transcription plasmid

[0037] Prepare the reaction system according to Table 1, slowly pipette and mix, centrifuge briefly, incubate at 37°C for 60 min in a PCR instrument, and then incubate at 65°C for 20 min for heat inactivation. After terminating the reaction, take a portion and run it through 1% agarose gel electrophoresis to compare whether the sequence size is correct. Then use a DNA purification kit to purify and recover the enzyme digestion product to obtain an in vitro transcription linearized template, quantify the DNA template, and store it at -20°C for later use.

[0038] Table 1 In vitro transcription plasmid linearization reaction system

[0039] Components Mass / Volume <![CDATA[pGEM-UTR-NP EBOV ]]> 1 μg 10×CutSmart buffer 5μL XbaⅠ 1μL Nuclease-Free Water To 50μL

[0040] 2) In vitro transcription

[0041] Use the linearized plasmid pGEM-UTR-NP EBOV As a template, replace uridine-5′-triphosphate (UTP) with N1-methyl-pseudouridine-5′-triphosphate (m1ΨTP) to produce mRNA in vitro by T7 RNA polymerase-mediated transcription. Set up the following 20 μL in vitro transcription reaction system on ice:

[0042] Table 2 In vitro transcription reaction system

[0043]

[0044]

[0045] Gently mix all components, centrifuge briefly, collect and incubate in a PCR instrument at 37℃ for 3h. Then add 1μL of DNase I to the reaction system and incubate at 37℃ for 15min to digest the DNA template. Transfer 20μL of in vitro transcription product to a 1.5mL centrifuge tube, add 30μL of 7.5M LiCl and 30μL of RNase Free Water respectively; after mixing, place at -20℃ for at least 30min. Then centrifuge at 12000rpm for 15min, remove the supernatant and collect the precipitate. Wash three times with pre-cooled 70% ethanol, and detect the RNA concentration after re-dissolving with RNase Free Water.

[0046] 3) mRNA capping and purification

[0047] Dilute 25 μg RNA to 33.5 μL with RNase-free water, heat at 65°C for 5 min, place on ice for 5 min, and add the following components in sequence to establish a 50 μL capping reaction system:

[0048] Table 3 Capping reaction system

[0049] Components Mass / Volume Denatured RNA 33.5μL 10×Capping Reaction Buffer 5μL GTP(10mM) 5μL SAM(20mM) 1.25μL Recombinant RNase Inhibitor(40U / μL) 1.25μL mRNA Cap 2′-O-Methyltransferase(100U / μL) 2μL Vaccinia Capping Enzyme(10U / μL) 2μL

[0050] Gently mix all components, centrifuge briefly, collect and incubate in a PCR instrument at 37℃ for 30min. Transfer 50μL of capped product to a 1.5mL centrifuge tube, add 75μL of 7.5M LiCl and 75μL of RNase Free Water respectively; after mixing, place at -20℃ for at least 30min. Then centrifuge at 12000rpm for 15min, remove the supernatant and collect the precipitate. Wash three times with pre-cooled 70% ethanol, and detect the RNA concentration after re-dissolving with RNase Free Water. NPEBOV The mRNA microfluidic capillary electrophoresis profile showed a single peak around 3000 nt ( Figure 2 ). And according to the capillary electrophoresis test, its purity was 99%, indicating the integrity and high purity of mRNA.

[0051] In the embodiment, we developed a vaccine platform based on mRNA encapsulated in LNP for in vivo delivery. The specific operation is as follows: lipids, including ALC-0315, DSPC, cholesterol and DMG-PEG 2000, are dissolved in ethanol at a molar ratio of 55:10:32.5:2.5 to prepare a 16mM lipid-ethanol mixture. Prepare mRNA working solution in citric acid buffer. Assemble the microfluidic hybrid chip, select syringes of corresponding volumes to inhale citric acid buffer and anhydrous ethanol respectively, and exhaust the air in the syringe. Select the cleaning mode to pre-rinse the pipeline and chip. After the pre-rinsing is completed, select syringes of corresponding volumes to inhale mRNA-citric acid buffer and lipid-ethanol mixture respectively, and exhaust the air in the syringe. Insert the sample and waste liquid collection tubes into their respective tube grooves. Adjust the total preparation volume according to the preparation volume, and select the appropriate syringe and waste liquid volume. Taking the total preparation volume of 2mL as an example, the flow rate ratio is set to 3:1, the total flow rate is set to 12mL / min, the left 2.5mL syringe, and the right 1mL syringe. At the same time, turn on the automatic waste liquid collection switch, discard 0.3mL of waste liquid before starting collection, and discard 0.05mL of waste liquid before the end of the operation. After sample collection, immediately dilute the sample collection liquid with 5 times the volume of PBS. After filtering through a 0.22μm filter, use a 30KD ultrafiltration tube and centrifuge at 4°C and 3000rpm for ultrafiltration until the solution is concentrated to less than 1mL. Dilute 5 times with PBS again and concentrate to less than 1mL. After ultrafiltration, the candidate nucleic acid vaccine mRNA-LNP was stored in a PBS solution containing 10% sucrose and stored at -80°C. LNP characterization was measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively.

[0052] The vaccine candidate mRNA (EBOV NP)-LNP manufactured based on the microfluidic strategy showed an average particle size of 64.21 nm ( Figure 3 ), the polydispersity coefficient is 0.04, and the encapsulation efficiency is more than 75%. Figure 4 Transmission electron microscopy (TEM) analysis showed that the mRNA-LNP particles all exhibited a uniform solid spherical morphology.

[0053] As shown in SEQ ID NO.1:

[0054]

[0055] SEQ ID NO.2:

[0056] ATMDSRPQKVWMTPSLTESDMDYHKILTAGLSVQQGIVRQRVIPVYQVNNLEEICQLIIQAFEAGVDFQESADSFLLMLCLHHAYQGDYKLFLESGAVKYLEGHGFRFEVKKRDGVKRLEELLPAVSSGRNIKRTLAAMPEEETTEANAGQFLSFASLFLPKLVVGEKACLEKVQRQIQVHAEQGLIQYPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVISNSVAQARFSGLLIVKTVLDHILQKTERGVRLHPLARTAKVKNEVNSFKAALSSLAKHGEYAPFARLLNLSGVNNLEHGLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQQYAESRELDHLGLDDQEKKILMNFHQKKNEISFQQTNAMVTLRKERLAKLTEAITAASLPKTSGHYDDDDDIPFPGPINDDDNPGHQDDDPTDSQDTTIPDVVVDPDDGGYGEYQSYSENGMSAPDDLVLFDLDEDDEDTKPVPNRSTKGGQQKNSQKGQHTEGRQTQSTPTQNVTGPRRTIHHASAPLTDNDRRNEPSGSTSPRMLTPINEEADPLDDADDETSSLPPLESDDEEQDRDGTSNRTPTVAPPAPVYRDHSEKKELPQDEQQDQDHIQEARNQDSDNTQPEHSFEEMYRHILRSQGPFDAVLYYHMMKDEPVVFSTSDGKEYTYPDSLEEEYPPWLTEKEAMNDENRFVTLDGQQFYWPVMNHRNKFMAILQHHQVDPGVDDYKDDDDKVDDYKDDDDKVDDYKDDDDK。

[0057] SEQ ID NO.3:

[0058]

[0059] Example 2

[0060] Immunization regimen

[0061] Six-week-old female BALB / c mice were randomly divided into groups. Each group received three intramuscular injections of candidate vaccines under isoflurane anesthesia on days 1, 28, and 84 to evaluate the immediate immune response of each group of candidate vaccines. The candidate nucleic acid vaccines for EBOV NP were: 30 μg of endotoxin-free DNA plasmid pVAX1-NP EBOV (NP D ) (n=8) and LNP-encapsulated 10 μg NP EBOV mRNA(NP R )(n=8) At the same time, for the above-mentioned candidate nucleic acid vaccines, PBS (n=8) was set as a control. Each candidate vaccine was injected intramuscularly. Serum samples were collected from the mouse eyeball blood on the 108th day after the initial immunization to detect neutralizing antibody responses. In addition, serum samples were collected from the mouse tail vein blood on the 35th and 98th days after the initial immunization to evaluate specific antibody responses. Spleen tissues of each group of immunized mice were collected on the 108th day after the initial immunization, and universal ELISpot and flow cytometry were used to evaluate cellular immune responses. During the observation period after immunization, no local inflammatory reactions or other adverse reactions were observed at the injection site.

[0062] Example 3

[0063] Nucleic acid vaccine induces the production of specific neutralizing antibodies in mice

[0064] The titer of EBOV NP-specific IgG antibody was determined by ELISA. The specific operation was as follows: the prokaryotic expression EBOV NP protein with a final concentration of 10 μg / mL was used for coating of 96-well ELISA plates and incubated at 4°C overnight. After coating, the liquid in the wells was shaken off and washed once with PBST; ELISA blocking solution was added, 200 μL / well, and blocked at 37°C for 40 min. The liquid in the wells was discarded, and the mouse serum was diluted with ELISA diluent in a multiple gradient from 1:100, and 100 μL of each dilution was added to the wells as the primary antibody and incubated at 37°C for 1 hour. Then the plate was washed five times, and each well received 100 μL of HRP-conjugated goat anti-mouse IgG diluted 1:5000 as the secondary antibody, and incubated at 37°C for 30 min. The secondary antibody solution was discarded, washed 6 times, and 100 μL / well TMB substrate was added, and incubated at room temperature in the dark for 5-30 min. Add 50 μL / well ELISA stop solution to terminate the reaction. Use an ELISA reader to measure the OD value of each well at a wavelength of 450 nm. The antibody titer is defined as the absorbance greater than the OD value of the PBS group. 450 The value is 2.1 times the serum dilution.

[0065] Blood was collected two weeks after each immunization and the production of NP-specific IgG antibodies in the serum of immunized mice was measured by ELISA. Figure 5 As shown, after each immunization, both candidate vaccine groups successfully induced the production of antigen-specific IgG antibodies.

[0066] Example 4

[0067] Nucleic acid vaccine induces specific T cell responses in mice

[0068] We used ELISpot to detect IFN-γ-secreting T cells in the spleen tissue of immunized mice 3 to 4 weeks after the booster injection to evaluate whether each group of immunization activated the EBOV antigen-specific T cell immune response, and stimulated the spleen cells of immunized mice with specific 9-peptides. Single spleen cells of mice were prepared under sterile conditions, and the specific cellular immune response of immunized mice was evaluated using the mouse IFN-γ ELISpot kit. The 9-peptide is closer to the short peptide fragment presented by MHC class I molecules after endogenous processing, and mainly targets CD8 + Recognition and activation of T cells. Therefore, the immune response under antigen-specific 9-peptide stimulation can indicate CD8 + Activation of T cell responses.

[0069] The results are as follows Figure 6 As shown, under the stimulation of NP 9-peptide mixture, the virus-specific T lymphocytes secreting IFN-γ in each group increased significantly compared with the control group, indicating that the antiviral Th1 type cellular immune response was successfully induced.

[0070] Example 5

[0071] Preliminary safety evaluation of nucleic acid vaccines

[0072] Three weeks after the last booster vaccination, the muscle tissue and major organs of the mice at the injection site were collected for H&E staining to preliminarily evaluate the safety of the vaccine. Figure 7 As shown, no significant pathological differences were observed in the gastrocnemius muscle, heart, liver, kidney, spleen, lung, and brain. These results confirmed that immunization of mice with the EBOV NP candidate vaccine did not cause tissue damage, supported the biocompatibility and safety of the vaccine on the major organs of mice, and provided preliminary evidence for the safety of the vaccine.

[0073] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0074] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A gene encoding the nucleocapsid protein of Zaire Ebola virus, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A vector containing the gene according to claim 1.

3. The carrier according to claim 2, characterized in that The vector is a pVAX1 vector.

4. Use of the vector according to any one of claims 2 to 3 in the preparation of a DNA vaccine for preventing Ebola virus.

5. A DNA vaccine for preventing Ebola virus, characterized in that: Containing the carrier according to any one of claims 2 to 3.

6. An mRNA molecule expressing the nucleocapsid protein of Zaire Ebola virus, characterized in that: The transcription template DNA of the mRNA molecule is formed by sequentially connecting the 5′UTR region, the gene encoding the Zaire Ebola virus nucleocapsid protein according to claim 1, the 3′UTR region and the PolyA tail; The mRNA molecule comprises a modified nucleoside; the modified nucleoside is a pseudouridine, the pseudouridine is N1-methyl-pseudouridine-5′-triphosphate, and all uridines in the mRNA molecule are replaced by N1-methyl-pseudouridine-5′-triphosphate.

7. Use of the mRNA molecule expressing the Zaire Ebola virus nucleocapsid protein according to claim 6 in the preparation of an mRNA vaccine for preventing Ebola virus.

8. An mRNA vaccine for preventing Ebola virus, characterized in that: It comprises the mRNA molecule expressing the Zaire Ebola virus nucleocapsid protein as claimed in claim 6 and at least one pharmaceutically acceptable excipient.

9. The mRNA vaccine for preventing Ebola virus according to claim 8, characterized in that: The mRNA molecules are formulated into lipid nanoparticles.

10. The method for preparing the mRNA vaccine for preventing Ebola virus according to claim 9, characterized in that: The mRNA molecule expressing the nucleocapsid protein of Zaire Ebola virus, ALC-0315, DSPC, cholesterol and DMG-PEG 2000 are mixed to prepare lipid nanoparticles to obtain the mRNA vaccine.

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