A novel rabies virus-like nucleocapsid nanostructure vaccine and its construction method

Abstract

The present invention relates to a novel rabies virus-like nucleocapsid nanostructure vaccine and a method for constructing the same, belonging to the field of biomedicine. The nanostructure vaccine of the present invention is formed by binding mRNA highly expressing rabies virus G protein and recombinant rabies virus N protein expressed by Escherichia coli to form an NLS structure as a vaccine antigen component, and then encapsulated with a lipid component. The vaccine constructed by the present invention can form an immunogenic signal stimulation integrated by G antigen and N antigen, enabling specific immunity to form effective responses in both humoral immunity and cellular immunity. In the challenge protection experiment, the vaccine can achieve 100% protection effect on mice.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine. Specifically, it relates to a novel rabies virus-like nucleocapsid nanostructure vaccine and a method for constructing the same. Background Art

[0002] Rabies virus belongs to the genus Lyssavirus in the family Rhabdoviridae, and its morphology is typically bullet-shaped, about 180 nm in length and about 80 nm in width. This virus is a single-stranded negative-sense RNA virus, with a genome of about 12,000 nt in length. Its structure mainly includes G protein, M protein, P protein, L protein, N protein, and viral RNA encapsulated by N protein.

[0003] This virus is a zoonotic pathogen. Animals carrying rabies virus, such as cats and dogs, will transmit the virus to humans after biting them. So far, rabies cases have occurred in more than 150 countries around the world. The fatality rate of this disease is close to 100%. The annual death toll due to this disease is 59,000, and about 40% are children under 14 years old. Although there is no effective drug for treating this disease, pre-exposure prophylactic vaccination and timely post-exposure vaccination with rabies vaccine can both effectively prevent the occurrence of this disease. Therefore, rabies vaccine is crucial for preventing rabies.

[0004] The existing rabies vaccines mainly include chicken embryo, hamster kidney cell, diploid cell, and Vero cell inactivated vaccines. However, the main drawbacks of the existing vaccines are low yield and low antibody titer after injection. Therefore, prophylactic immunization requires 3 injections, while post-exposure immunization requires 4 or 5 injections. To overcome the problems of the existing vaccines, many teams have also made a lot of explorations in the research and development of novel rabies vaccines. Some teams have successfully expressed rabies virus G protein as a vaccine candidate antigen through yeast, HEK 293 cells, BHK-21 cells, CHO cells, insect cells, and even plant cells. However, more research and technical support are still needed for it to be marketed as a vaccine. There are also those who have constructed virus-like particles (VLPs) of rabies virus through the combination of G protein and M protein. After immunizing mice and conducting a rabies virus challenge experiment, it was confirmed that it had a 90% protective effect on mice, but it did not achieve a complete protective effect. Therefore, a more efficient rabies vaccine is needed to replace the existing vaccines. Summary of the Invention

[0005] In view of the problems existing in the background art, the present invention provides a novel virus nucleocapsid-like nanostructure (NLS) vaccine and its preparation method. The vaccine is formed by binding mRNA highly expressing rabies virus G protein and recombinant rabies virus N protein expressed by Escherichia coli to form an NLS structure as the vaccine antigen component, and then encapsulated with a lipid component. The vaccine constructed by the present invention can induce stronger B cell and T cell immune responses, and the mouse challenge test also confirms that the NLS vaccine can achieve a 100% protection effect.

[0006] To achieve the above object, the present invention is realized through the following technical solutions:

[0007] The novel rabies virus nucleocapsid-like nanostructure vaccine (rabies virus NLS vaccine) includes an mRNA molecular structure expressing rabies virus G protein antigen; the mRNA molecular structure expressing rabies virus G protein antigen includes: a flavivirus 5' untranslated region gene fragment, an Igκ secretion signal sequence, a rabies virus G protein sequence, a flavivirus 3' UTR gene fragment, and a polyA tail;

[0008] The sequence of the flavivirus 5' UTR is as shown in SEQ ID NO.1; the sequence of the Igκ secretion signal is as shown in SEQ ID NO.2; the sequence of the rabies virus G protein is as shown in SEQ ID NO.3; the sequence of the flavivirus 3' UTR is as shown in SEQ ID NO.4; the sequence of the polyA tail is as shown in SEQ ID NO.5.

[0009] The novel rabies virus nucleocapsid-like nanostructure vaccine is an mRNA-protein complex structure formed by the binding of an mRNA molecule expressing G protein antigen and an N protein molecule.

[0010] Furthermore, the N protein is a recombinant rabies virus N protein expressed by Escherichia coli.

[0011] Furthermore, the method for obtaining the N protein is as follows: Insert the 6×His sequence CACCACCACCACCACCAC and the nucleic acid sequence SEQ ID NO.6 expressing rabies virus N protein into the pET30a vector, and name the recombinant plasmid pET30a-RABV-N. Transform the recombinant plasmid into BL21(DE3) competent Escherichia coli, select monoclonal colonies and perform fermentation. When the OD of the bacterial liquid 600 = 0.8, add 0.1 mM IPTG and induce at 16°C for 24 h. Harvest the bacterial cells, break the bacterial cells and centrifuge to collect the supernatant, and obtain the recombinant rabies virus N protein through His-tag affinity chromatography.

[0012] The construction method of the novel rabies virus-like nucleocapsid nanostructure vaccine comprises the following steps:

[0013] (1) The mRNA expressing the rabies virus G protein antigen and the recombinant rabies virus N protein expressed by Escherichia coli are formulated into an aqueous phase at a mass ratio of 4:1, and the following buffer system is added: 20 mM NaAc, 10 mM MgCl 2 10 mM, 2 mM DTT, 50 mM NaCl (all are the final concentrations of the system), and react at 37 °C for 1 h to form a nucleocapsid-like structure;

[0014] (2) Prepare the NLS vaccine lipid delivery system: ((2-(2-hydroxyethoxy)ethyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (DHA-1), 1,2-dioleoyl-3-trimethylammonium-chloride (DOTAP-Cl), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and methoxypolyethylene glycol-N-tetradecyl-tetradecanamide (mPEG-DTA-1-2K) with a molar ratio of 36-40:6-10:16-20:0.8-1.2; respectively add 1.0%-1.4% sorbitan trioleate Span 85 and 0.4%-0.6% polyoxyethylene sorbitan monooleate Tween-80 as lipid stabilizers;

[0015] (3) Encapsulation: Homogenize the prepared lipid phase and aqueous phase to form lipid nanoparticles with a TRR = 1:3 and a TFR = 100 ml / min.

[0016] (4) The encapsulated lipid nanoparticles are diluted with a buffer containing 10 mM NaAc and 0.001% trehalose at pH = 6.4 by 30-35 times the volume, concentrated through a 100 kDa filter membrane, and then diluted with a solution of 20 mM NaAc, 0.01% trehalose, and 3.5% sucrose, and filtered and sterilized to obtain the NLS vaccine.

[0017] The beneficial effects of the present invention:

[0018] The novel rabies virus-like nucleocapsid nanostructure vaccine of the present invention does not use simple mRNA that can express the rabies virus G protein as a vaccine component. Instead, it uses mRNA that highly expresses the rabies virus G protein and the recombinant rabies virus N protein expressed by Escherichia coli BL21(DE3) to form an NLS structure as the vaccine antigen component, and then constructs a novel composite structure vaccine by encapsulating it with lipid components. The first reason for this design is that the G protein is the most important antigen of the rabies virus. In addition to inducing the body to produce specific antibodies, it can also effectively induce T cell immunity; the N protein is also an effective protective antigen, which can stimulate B cells and T cells to produce humoral immunity and cellular immunity; in addition, in natural rabies virus, the N protein and RNA itself are combined as a complex; the second reason is that through our experiments, it is confirmed that compared with the simple mRNA vaccine without N protein, the NLS vaccine with N protein can induce stronger B cell and T cell immune responses, and the mouse challenge experiment also confirms that the NLS vaccine can achieve 100% protection effect. Brief Description of the Drawings

[0019] Figure 1 It is a schematic diagram of the mRNA molecular structure expressing the rabies virus G protein antigen of the novel rabies virus-like nucleocapsid nanostructure vaccine of the present invention;

[0020] Figure 2 It is the expression effect of the mRNA expressing the rabies virus G protein antigen transfected into 293T cells in Example 2 of the present invention;

[0021] Figure 3 It is the SDS-PAGE electrophoresis and silver staining results after purification of the recombinant rabies virus N protein;

[0022] Figure 4 It is an electron micrograph of the nucleocapsid-like structure;

[0023] Figure 5 It is an electron micrograph of the novel rabies virus-like nucleocapsid nanostructure vaccine of the present invention;

[0024] Figure 6 It is the result of the specific antibodies and cellular immunity of the rabies virus G protein and N protein induced by the NLS vaccine of the present invention;

[0025] Figure 7 It is the survival curve of the mouse challenge experiment of the NLS vaccine of the present invention. Detailed Description of the Invention

[0026] To make the objectives, technical solutions and beneficial effects of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts all fall within the scope of protection of the present invention.

[0027] To illustrate the present invention more clearly, the following embodiments are used for detailed description. Embodiment 1

[0028] A novel rabies virus-like nucleocapsid nanostructure vaccine, which is a mRNA-protein complex structure formed by the binding of mRNA molecules expressing G protein antigen and N protein molecules (the molecular structure is as shown in the appendix Figure 1 ).

[0029] Among them, the mRNA molecular structure expressing rabies virus G protein antigen includes: flavivirus 5'-untranslated region gene fragment (gene sequence as SEQ ID NO.1), Igκ secretion signal sequence (gene sequence as SEQ ID NO.2), rabies virus G protein (gene sequence as SEQ ID NO.3), flavivirus 3' UTR gene fragment (gene sequence as SEQ ID NO.4) and polyA tail (gene sequence as SEQ ID NO.5).

[0030] The N protein is a recombinant rabies virus N protein expressed by Escherichia coli. Embodiment 2

[0031] The mRNA molecular structure expressing rabies virus G protein antigen as in Example 1 is constructed as follows: The flavivirus 5'-untranslated region gene fragment (gene sequence as SEQ ID NO.1), Igκ secretion signal (gene sequence as SEQ ID NO.2), rabies virus G protein (gene sequence as SEQ ID NO.3), flavivirus 3' UTR gene fragment (gene sequence as SEQ ID NO.4), and polyA tail (gene sequence as SEQ ID NO.5) are inserted into the pVAX1 vector through Hind III and Bam H I restriction enzyme sites to obtain the mRNA template plasmid. Using the BamH I-linearized plasmid as the template, in vitro transcription is carried out by T7 enzyme (system: 2 μl of NEB 10X T7 CleanCap Reagent AG Reaction Buffer, 2 μl of NEB ATP (60 mM), 2 μl of NEB GTP (50 mM), 1 μl of methylated N1-Me-pUTP (100 mM), 2 μl of NEB CTP (50 mM), 2 μl of NEB CleanCap Reagent AG (40 mM), 1 μg of template DNA, 2 μl of NEB T7 RNA Polymerase Mix, make up to 20 μl with enzyme-free water; reaction conditions: react at 37°C for 2 h; add 2 μl of NEB DNase I and react at 37°C for 15 min to remove the DNA template) to prepare G-mRNA. After mixing this mRNA with the transfection reagent Lipofectin, it is transfected into 293T cells (mix 5 μl of Novoprotein lipomaster 2000 and 2.5 μg of mRNA and transfect a 6-well plate, and at the same time set a blank control well with only the transfection reagent and no mRNA), and the expression of the G protein antigen can be obtained clearly (attached Figure 2 ).

[0032] The recombinant rabies virus N protein expressed by Escherichia coli in Example 1 was obtained by inserting the 6×His sequence CACCACCACCACCACCAC and the nucleic acid sequence expressing the rabies virus N protein (such as SEQ ID NO.7) into the pET30a vector (method: adding 1 μg of the target gene and the vector to 1 μl of restriction enzymes, NEB Nde I and Hind III restriction enzymes, double-digesting the target gene and the vector and inserting them). The recombinant plasmid was named pET30a-RABV-N. The recombinant plasmid was transformed into BL21(DE3) competent Escherichia coli (the competent cells were thawed on ice for 20 minutes, 1 ng of the recombinant plasmid was added, placed on ice for 30 minutes, heat-shocked at 42 °C for 90 seconds, placed on ice for 2 minutes, added with LB medium and resuscitated at 37 °C for 1 hour, and 200 μl was taken and spread on an LB plate containing 50 μg / ml kanamycin). Single colonies were selected and fermented (LB medium: containing 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl per liter of medium, adjusted to pH 7.0 with hydrochloric acid; conditions: the initial OD 600 value was 0.1 - 0.2, cultured at 30 °C and 150 rpm). When the OD 600 of the bacterial liquid reached 0.8, 0.1 mM IPTG was added and induced at 16 °C for 24 h. The bacteria were harvested, and after disrupting the bacteria, the supernatant was collected by centrifugation. The recombinant rabies virus N protein with a purity of over 90% was obtained by His-tag affinity chromatography (attached Figure 3 is the SDS-PAGE electrophoresis and silver staining results of the obtained recombinant rabies virus N protein). Example 3

[0033] The construction method of the rabies virus nucleocapsid-like structure (NLS) vaccine includes the following steps:

[0034] (1) Construction of the nucleocapsid-like structure

[0035] The mRNA expressing the rabies virus G protein antigen in Example 2 and the recombinant rabies virus N protein were formulated into an aqueous phase at a mass ratio of 4:1, and an acetate buffer system: NaAc 20 mM, pH 5.0, MgCl 2 10 mM, DTT 2 mM, NaCl 50 mM was added, and reacted at 37 °C for 1 h to form a nucleocapsid-like structure (electron micrograph is attached Figure 4 );

[0036] (2) Preparation of the lipid delivery system of the NLS (nucleocapsid-like structure) vaccine

[0037] Nanopaper particles: ((2-(2-hydroxyethoxy)ethyl)azaalkanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (DHA-1), 1,2-dioleoyl-3-trimethylammonium-chloride (DOTAP-Cl), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and methoxypolyethylene glycol-N-tetradecyl-tetradecanamide (mPEG-DTA-1-2K) with a molar ratio of 36-40:6-10:16-20:0.8-1.2; 1.0%-1.4% sorbitan trioleate Span 85 and 0.4%-0.6% polyoxyethylene sorbitan monooleate Tween-80 are respectively added as lipid stabilizers to obtain a lipid delivery system;

[0038] (3) Encapsulation: The lipid phase in step (2) and the nucleocapsid-like structure in step (1) are homogenized using a microfluidic device at a volume ratio of lipid:aqueous phase = 1:3 (nitrogen-phosphorus ratio is 4-10:1) to form lipid nanoparticles with a TRR = 1:3 and a TFR = 100 ml / min.

[0039] (4) The encapsulated lipid nanoparticles are diluted 30-35-fold with a buffer containing 10 mM NaAc and 0.001% trehalose, pH = 6.4, concentrated through a 100 kDa filter membrane, and then diluted with a solution of 20 mM NaAc, 0.01% trehalose and 3.5% sucrose, and filtered and sterilized to obtain the NLS vaccine (electron micrographs are as attached Figure 5 )

[0040] Verification of the immunological effect of the obtained rabies vaccine:

[0041] The rabies virus nucleocapsid-like structure vaccine established in this design shows better performance in its immunological evaluation than inactivated vaccines and mRNA vaccines expressing only the G protein.

[0042] Antibody detection and Elispot assay method: The encapsulated NLS vaccine (20 μg mRNA + 5 μg protein), pure mRNA vaccine (containing 20 μg mRNA), traditional inactivated vaccine and PBS blank control group are used to immunize Blab / c mice, a total of 4 groups, 5 mice in each group, 25 mice with half males and half females, randomly grouped. Immunize 3 doses at 0, 7, 21 d by intramuscular injection in the limbs. Collect blood from the tail vein at 7, 21, 35 d after the first immunization, separate the serum and measure the antibody titers of RABV-G and RABV-N by the Elisa method; and sacrifice after collecting blood at 35 d, and detect the expression level of IFN-γ cytokine by the Elispot method. The results show that the NLS vaccine can produce specific antibodies and cellular immunity higher than those of inactivated vaccines and mRNA vaccines encoding only the G antigen alone ( Figure 6 )

[0043] Mouse challenge protection experiment method: The encapsulated NLS vaccine (20 μg mRNA + 5 μg protein), the simple mRNA vaccine (containing 20 μg mRNA), the traditional inactivated vaccine and the PBS blank control group were used to immunize Kunming mice. There were 4 groups in total, with 20 mice in each group. Among the 80 mice, half were male and half were female, and they were randomly grouped. Immunize 3 doses on days 0, 7, and 21 by intramuscular injection in the four limbs. At 35 days after the first immunization, challenge with 25 LD through the footpad 50 , and record the death situation of the mice. The results showed that in the challenge protection experiment, the vaccine could achieve 100% protection effect on mice ( Figure 7 ).

[0044] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in terms of form and details without departing from the scope defined by the claims of the present invention.

[0045] Sequence number SEQ ID NO.1

[0046] Sequence name Gene fragment of flavivirus 5' UTR

[0047] Length: 124

[0048] Molecular type: DNA

[0049] agtaaatcct gtgtgctaat tgaggtgcat tggtctgcaa atcgagttgc taggcaataa 60

[0050] acacatttgg attaatttta atcgttcgtt gagcgattag cagagaactg accagaacgc 120

[0051] cacc

[0052] Sequence number SEQ ID NO.2

[0053] Sequence name Igκ secretion signal sequence

[0054] Length 60

[0055] Molecular type DNA

[0056] Organism synthetic construct

[0057] gagacagaca cactcctgct atgggtactg ctgctctggg ttccaggttc cactggtgac

[0058] Sequence number SEQ ID NO.3

[0059] Sequence name Rabies virus G protein sequence

[0060] Length 1317

[0061] Molecular type DNA

[0062] agcaagggct ctaaaacctg cggattcgtc gacgagcggg gcctgtacaa gtccctgaaa 660

[0063] ggcgcctgta aactgaagct gtgcggcgtg ctgggacttc ggctcatgga cggaacatgg 720

[0064] gtcgccatcc aaacaagcaa cgagacaaag tggtgccccc ccgatcagct ggttaatctg 780

[0065] catgatttcc acagcgacga gatcgagcac ctggtggtcg aagagctggt gaaaaagcgg 840

[0066] gaagagtgcc tggatgccct ggaaagcatc atgacaacca agtccgtgag ttttcggaga 900

[0067] ctgagccacc tgaggaagct cgtgcccggc ttcggcaagg cctacaccat tttcaacaag 960

[0068] accctgatgg aagccgacgc tcactacaag tccgttagaa cctggaacga gatcatccct 1020

[0069] agcaagggat gtctgagagt gggcggcaga tgccaccccc acgtgaacgg cgtgttcttc 1080

[0070] aatggcatca tcctgggccc tgatggacac gttctgatcc cagagatgca gagctctctg 1140

[0071] ctgcagcagc acatggaact gctggaaagc agcgtgatcc cactgatgca ccctctggcc 1200

[0072] gatcctagca ccgtgtttaa ggacggcgac gaggtggagg acttcgtgga agtgcacctg 1260

[0073] cctgacgtgc ataagcaggt gtccggcgtg gacctgggcc tgcctaattg gggaaaa 1317

[0074] Sequence number SEQ ID NO.4

[0075] Sequence name Flavivirus 3' UTR sequence

[0076] Length 123

[0077] Molecule type DNA

[0078] gacactgttc caagcaacaa ccaagaacta gaccttgtac ataggacaaa actgaaaccg 60

[0079] ggataaaaac cacggatgga gaaccggact ccacacatca aacagaagag gatgtcagcc 120

[0080] cag

[0081] Sequence number SEQ ID NO.5

[0082] Sequence name polyA tail

[0083] Length 138

[0084] Molecule type DNA

[0085] aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa gcatatgact aaaaaaaaaa 60

[0086] aaaaaaaaaa aaaaaaaaaa aaaaaaaacc gcgtgctgaa aaaaaaaaaa aaaaaaaaaa 120

[0087] aaaaaaaaaa aaaaaaaa

[0088] Sequence number SEQ ID NO.6

[0089] Sequence name Nucleic acid sequence expressing rabies virus N protein

[0090] Length 1347

[0091] Molecular type DNA

[0092] agcgccattc gggtgggcac cgtggtgacc gcctacgagg actgcagcgg ccttgtgtct 720

[0093] tttaccggct tcatcaagca gatcaacctg accgcccgag aagccatcct gtacttcttc 780

[0094] cacaagaact tcgaagagga gatcagaaga atgttcgagc ctggccaaga gacagctgtg 840

[0095] ccccacagct attttatcca ctttagaagc ctgggcctga gcggaaagtc cccatactct 900

[0096] agcaatgccg tgggccacgt gttcaacctg atccacttcg tgggctgcta catgggccaa 960

[0097] gtgcggagcc tgaacgctac agtgatcgct gcctgcgccc ctcacgagat gagcgtgctg 1020

[0098] ggaggttatc tgggcgagga attcttcggc aaaggcacct tcgagagacg gtttttccgg 1080

[0099] gatgagaagg aactgcagga gtacgaggcc gctgagctga caaagtctga tgtggccctg 1140

[0100] gccgacgacg gcacagtgaa cagcgacgac gaggactact tcagcggcga aaccagatct 1200

[0101] ccagaggcag tgtacacacg gatcatgatg aacggtggaa gactgaagag aagccacatc 1260

[0102] agacggtacg tgtccgtgtc cagcaaccac caggccagac ctaattcttt cgccgagttc 1320

[0103] ctgaacaaga cctacagcaa cgacagc 1347

Claims

1. A novel rabies virus nucleocapsid nanostructure vaccine, characterized in that: The novel rabies virus nucleocapsid nanostructure vaccine comprises an mRNA molecule expressing a rabies virus G protein antigen combined with a recombinant rabies virus N protein molecule to form an mRNA-protein complex structure, and the mRNA-protein complex structure is encapsulated by lipid nanoparticles to form a nucleocapsid nanostructure vaccine; The mRNA molecular structure expressing the rabies virus G protein antigen comprises: a gene fragment of the 5' non-coding region of the flavivirus, a Kozak sequence, an Igκ secretion signal sequence, a rabies virus G protein sequence, a 3' UTR gene fragment of the flavivirus and a polyA tail; The sequence of the flavivirus 5'UTR is as shown in SEQ ID NO.1; the sequence of the Igκ secretion signal is as shown in SEQ ID NO.2; the Kozak sequence is GCCACC; the mRNA sequence of the rabies virus G protein is as shown in SEQ ID NO.3; the sequence of the flavivirus 3'UTR is as shown in SEQ ID NO.4; and the sequence of the polyA tail is as shown in SEQ ID NO.

5.

2. The novel rabies virus nucleocapsid nanostructure vaccine according to claim 1, characterized in that: The N protein is the recombinant rabies virus N protein expressed by Escherichia coli.

3. The novel rabies virus nucleocapsid nanostructure vaccine according to claim 1 or 2, characterized in that: The method for obtaining the N protein is as follows: inserting the 6×His sequence CACCACCACCACCACCAC and the nucleic acid sequence SEQ ID NO.6 expressing the rabies virus N protein into the pET30a vector, naming the recombinant plasmid pET30a-RABV-N, transforming the recombinant plasmid into BL21 (DE3) competent Escherichia coli, selecting a single clone and fermenting until the bacterial liquid OD 600 =0.8, 0.1 mM IPTG was added and induced at 16°C for 24 h, the bacteria were harvested, the bacteria were broken and centrifuged to collect the supernatant, and the recombinant rabies virus N protein was obtained by His tag affinity chromatography.

4. The novel rabies virus nucleocapsid nanostructure vaccine according to claim 1, characterized in that: The lipid nanoparticles include ((2-(2-hydroxyethoxy)ethyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (DHA-1), 1,2-dioleoyl-3-trimethylamine-chloride (DOTAP-Cl), 1,2-dioleoyl-SN-glycero-3-phosphocholine (DOPC) and methoxy polyethylene glycol-N-tetradecyl-tetradecanoamide (mPEG-DTA-1-2K) in a molar ratio of 36-40:6-10:16-20:0.8-1.2; and 1.0%-1.4% of sorbitan trioleate Span 85 and 0.4%-0.6% of polyoxyethylene sorbitan monooleate Tween-80.

5. The novel rabies virus nucleocapsid nanostructure vaccine according to any one of claims 1 to 4, wherein the method for constructing the vaccine comprises the following steps: (1) mRNA expressing rabies virus G protein antigen and recombinant rabies virus N protein expressed by Escherichia coli were prepared into an aqueous phase at a mass ratio of 4:1, and the following buffer system was added: NaAc final concentration 20 mM, MgCl2 final concentration 10 mM, DTT final concentration 2 mM, NaCl final concentration 50 mM, and reacted at 37°C for 1 h to form a nucleocapsid-like structure; (2) Preparation of NLS vaccine lipid delivery system: ((2-(2-hydroxyethoxy)ethyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (DHA-1), 1,2-dioleoyl-3-trimethylamine-chloride (DOTAP-Cl), 1,2-dioleoyl-SN-glycero-3-phosphocholine (DOPC) and methoxypolyethylene glycol-N-tetradecyl-tetradecanoamide (mPEG-DTA-1-2K) in a molar ratio of 36-40:6-10:16-20:0.8-1.2; 1.0%-1.4% of sorbitan trioleate Span85 and 0.4%-0.6% of polyoxyethylene sorbitan monooleate Tween-80 were added as lipid stabilizers respectively; (3) Encapsulation: The prepared lipid phase and aqueous phase were homogenized to form lipid nanoparticles with TRR = 1:3 and TFR = 100 ml / min; (4) The encapsulated lipid nanoparticles were diluted 30-35 times with a buffer solution containing 10 mM NaAc, 0.001% trehalose, pH = 6.4, concentrated with a 100 kDa filter membrane, and then diluted with a 20 mM NaAc, 0.01% trehalose and 3.5% sucrose solution. After filtration and sterilization, the novel rabies virus nucleocapsid nanostructured vaccine was obtained.

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