Novel rabies virus nucleocapsid nanostructure vaccine and construction method thereof

Abstract

The invention relates to a novel rabies virus nucleocapsid nanostructure vaccine and a construction method thereof, and belongs to the field of biomedicine.The nanostructure vaccine is characterized in that mRNA capable of efficiently expressing rabies virus G protein and recombinant rabies virus N protein expressed by escherichia coli are combined to form an NLS structure serving as a vaccine antigen component; and then wrapping with a lipid component. The vaccine constructed by the invention can form G antigen and N antigen integrated immunogen signal stimulation, so that specific immunity can form an effective reaction in humoral immunity and cellular immunity. In a protective test of challenge, the vaccine can achieve a 100% protection effect on mice.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically, it relates to a novel rabies virus nucleocapsid nanostructure vaccine and its construction method. Background Technology

[0002] Rabies virus belongs to the genus *Rabiesvirus* in the family Rhabdoviridae. It has a typical bullet-shaped morphology, approximately 180 nm long and 80 nm wide. This virus is a single-stranded negative-sense RNA virus with a genome length of approximately 12,000 nt. Its main structures include G protein, M protein, P protein, L protein, N protein, and viral RNA encapsulated by the N protein.

[0003] Rabies is a zoonotic pathogen; animals carrying the rabies virus, such as cats and dogs, can transmit the virus to humans through bites. To date, cases of rabies have been reported in more than 150 countries worldwide. The disease has a near 100% fatality rate, causing 59,000 deaths annually, with approximately 40% of these deaths occurring in children under the age of 14. Although there is no effective treatment for rabies, pre-exposure prophylaxis and timely post-exposure rabies vaccination are highly effective in preventing the disease. Therefore, rabies vaccination is crucial in preventing rabies.

[0004] Existing rabies vaccines mainly include inactivated vaccines derived from chicken embryos, hamster kidney cells, diploid cells, and Vero cells. However, the main drawbacks of existing vaccines are low yield and low antibody titers after injection. Therefore, prophylactic immunization requires three doses, while post-exposure immunization requires four or five doses. To overcome the problems of existing vaccines, many teams have explored the development of novel rabies vaccines. Some teams have successfully expressed rabies virus G protein as a vaccine candidate antigen using yeast, HEK 293 cells, BHK-21 cells, CHO cells, insect cells, and even plant cells. However, further research and technical support are needed for these vaccines to be commercialized. Other teams have constructed virus-like particles (VLPs) of rabies virus by combining G and M proteins. Immunization of mice using rabies virus challenge experiments has shown a 90% protective effect, but not complete protection. Therefore, a more effective rabies vaccine is needed to replace existing vaccines. Summary of the Invention

[0005] To address the problems existing in the background art, this invention provides a novel viral nucleocapsid-like nanostructure (NLS) vaccine and its construction method. This vaccine uses an NLS structure formed by combining mRNA highly expressing rabies virus G protein and recombinant rabies virus N protein expressed by Escherichia coli as the vaccine antigen component, which is then encapsulated with a lipid component. The vaccine constructed by this invention can induce a stronger B cell and T cell immune response, and mouse challenge experiments have also confirmed that this NLS vaccine can achieve 100% protective efficacy.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: The novel rabies virus nucleocapsid 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' uncoding region gene fragment, an Igκ secretion signal sequence, a rabies virus G protein sequence, a flavivirus 3' UTR gene fragment, and a polyA tail. The sequence of the flavivirus 5' UTR is shown in SEQ ID NO.1; the sequence of the Igκ secretion signal is shown in SEQ ID NO.2; the sequence of the rabies virus G protein is shown in SEQ ID NO.3; the sequence of the flavivirus 3' UTR is shown in SEQ ID NO.4; and the sequence of the polyA tail is shown in SEQ ID NO.5.

[0007] The novel rabies virus nucleocapsid nanostructure vaccine is an mRNA-protein complex structure formed by the binding of an mRNA molecule expressing a G protein antigen and an N protein molecule.

[0008] Furthermore, the N protein is the recombinant rabies virus N protein expressed by Escherichia coli.

[0009] Furthermore, the method for obtaining the N protein is as follows: 6×His sequence CACCACCACCACCACCACCAC and the nucleic acid sequence SEQ ID NO.6 expressing the rabies virus N protein are inserted into the pET30a vector, and the recombinant plasmid is named pET30a-RABV-N. The recombinant plasmid is transformed into BL21(DE3) competent Escherichia coli, single clones are selected and fermented until the bacterial culture reaches OD. 600 When the concentration of the virus was 0.8, 0.1 mM IPTG was added and the virus was induced at 16 °C for 24 h. The bacterial cells were harvested, and after the bacterial cells were broken up, the supernatant was collected by centrifugation. The recombinant rabies virus N protein was obtained by His tag affinity chromatography.

[0010] The method for constructing the novel rabies virus nucleocapsid nanostructure vaccine includes the following steps: (1) The mRNA expressing rabies virus G protein antigen and the recombinant rabies virus N protein expressed by Escherichia coli were prepared into an aqueous phase at a mass ratio of 4:1. The following buffer system was added: NaAc 20mM, MgCl2 10mM, DTT 2mM, NaCl 50mM (all are final concentrations of the system). The reaction was carried out at 37℃ for 1h 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-glycerol-3-phosphocholine (DOPC) and methoxy polyethylene glycol-N-tetradecyltetradecylamide (mPEG-DTA-1-2K) were added in a molar ratio of 36-40:6-10:16-20:0.8-1.2; 1.0%-1.4% of sorbitan trioleate Span 85 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 are homogenized to form lipid nanoparticles with a TRR of 1:3 and a TFR of 100 ml / min.

[0011] (4) The encapsulated lipid nanoparticles were diluted 30 to 35 times by volume with a buffer containing 10 mM NaAc and 0.001% trehalose at pH 6.4, concentrated with a 100 kDa filter membrane, and then diluted with a solution of 20 mM NaAc, 0.01% trehalose and 3.5% sucrose. After filtration and sterilization, the NLS vaccine was obtained.

[0012] The beneficial effects of this invention are: This invention presents a novel rabies virus nucleocapsid nanostructure vaccine. Instead of using mRNA expressing the rabies virus G protein as a single vaccine component, it utilizes a novel composite structure constructed by combining highly efficient rabies virus G protein-expressing mRNA with recombinant rabies virus N protein expressed by Escherichia coli BL21(DE3) to form an NLS structure as the vaccine antigen component, which is then encapsulated by a lipid component. This design serves two main purposes: firstly, the G protein is the most important antigen of the rabies virus, inducing not only specific antibodies but also effective T-cell immunity; the N protein is also an effective protective antigen, stimulating humoral and cellular immunity in B and T cells; furthermore, in natural rabies virus, the N protein and RNA are inherently bound together as a complex. Secondly, our experiments have demonstrated that, compared to a simple mRNA vaccine without the N protein, the NLS vaccine with the N protein induces a stronger B-cell and T-cell immune response. Mouse challenge experiments have also confirmed that this NLS vaccine achieves 100% protective efficacy. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the mRNA molecular structure of the novel rabies virus nucleocapsid nanostructure vaccine of the present invention, expressing the rabies virus G protein antigen. Figure 2 This is the expression effect of mRNA expressing rabies virus G protein antigen transfected into 293T cells in Example 2 of the present invention; Figure 3 These are the results of SDS-PAGE electrophoresis and silver staining after purification of the recombinant rabies virus N protein; Figure 4 These are electron microscope images of nucleocapsid structures; Figure 5 This is an electron microscope image of the novel rabies virus nucleocapsid nanostructure vaccine of this invention; Figure 6 The results of rabies virus G and N protein-specific antibodies and cellular immunity induced by the NLS vaccine of this invention; Figure 7 This is the survival curve of the NLS vaccine of the present invention in a mouse challenge experiment. Detailed Implementation

[0014] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are all within the scope of protection of this invention.

[0015] To illustrate the present invention more clearly, the following embodiments will be described in detail. Example 1

[0016] A novel rabies virus nucleocapsid nanostructure vaccine, comprising an mRNA-protein complex structure formed by the binding of an mRNA molecule expressing the G protein antigen and an N protein molecule (molecular structure shown in the attached figure). Figure 1 ).

[0017] The mRNA molecular structure expressing the rabies virus G protein antigen includes: a gene fragment of the 5' uncoding region of flavivirus (gene sequence as SEQ ID NO.1), an Igκ secretion signal sequence (gene sequence as SEQ ID NO.2), a rabies virus G protein (gene sequence as SEQ ID NO.3), a flavivirus 3' UTR gene fragment (gene sequence as SEQ ID NO.4), and a polyA tail (gene sequence as SEQ ID NO.5).

[0018] The N protein is the recombinant rabies virus N protein expressed by Escherichia coli. Example 2

[0019] The mRNA molecule structure expressing rabies virus G protein antigen, as described in Example 1, is constructed as follows: the 5' uncoding region gene fragment of flavivirus (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 HI restriction sites to obtain the mRNA template plasmid. Using BamHI linearized plasmid as a template, G-mRNA was prepared by in vitro transcription with T7 enzyme (system: NEB 10XT7 CleanCap Reagent AG Reaction Buffer 2 μl, NEB ATP (60 mM) 2 μl, NEB GTP (50 mM) 2 μl, NEB N1-Me-pUTP (100 mM) 1 μl, NEB CTP (50 mM) 2 μl, NEB CleanCapReagent AG (40 mM) 2 μl, template DNA 1 μg, NEB T7 RNA Polymerase Mix 2 μl, and enzyme-free water to 20 μl; reaction conditions: 37℃ for 2 h; 2 μl NEB DNase I was added, and the DNA template was removed by reaction at 37℃ for 15 min). The mRNA was mixed with the transfection reagent Lipofectin and transfected into 293T cells (2000 5 μl of Novizan Lipomaster and 2.5 μg of mRNA were mixed and transfected into 6-well plates; blank control wells containing only the transfection reagent and no mRNA were also included). Clear expression of the G protein antigen was obtained (see attached). Figure 2 ).

[0020] The recombinant rabies virus N protein expressed by *Escherichia coli* in Example 1 was obtained by inserting a 6×His sequence CACCACCACCACCACCACCAC and the nucleic acid sequence expressing the rabies virus N protein (e.g., SEQ ID NO. 7) into the pET30a vector (method: 1 μg of the target gene and 1 μl of the vector were added to NEB Nde I and Hind III restriction endonucleases for double digestion of the target gene and vector insertion). The recombinant plasmid was named pET30a-RABV-N. The recombinant plasmid was transformed into BL21(DE3) competent *Escherichia coli* (competent cells were thawed on ice for 20 minutes, 1 ng of the recombinant plasmid was added, the cells were placed on ice for 30 minutes, heat-shocked at 42°C for 90 seconds, placed on ice for 2 minutes, and then thawed at 37°C for 1 hour in LB medium. 200 μl of the thawed plasmid was then plated on LB plates containing 50 μg / ml kanamycin). Single clones were selected and fermented (LB medium: each liter of medium contained 10 g trypsin, 5 g yeast extract, and NaCl). 10g, adjust pH to 7.0 with hydrochloric acid; conditions: initial OD of inoculated strain. 600 Value is 0.1~0.2 (cultured at 30℃ and 150rpm), fermentation until the OD of the bacterial culture is reached. 600 When the concentration of the virus was 0.8, 0.1 mM IPTG was added and induced at 16°C for 24 h. The bacterial cells were harvested, lysed, and the supernatant was collected by centrifugation. Recombinant rabies virus N protein with a purity of over 90% was obtained by His-tag affinity chromatography (see attached image). Figure 3 (SDS-PAGE electrophoresis and silver staining results of the obtained recombinant rabies virus N protein). Example 3

[0021] A method for constructing a rabies virus nucleocapsid-like (NLS) vaccine includes the following steps: (1) Components of a nucleocapsid structure The mRNA expressing rabies virus G protein antigen from Example 2 and the recombinant rabies virus N protein were prepared into an aqueous phase at a mass ratio of 4:1. A sodium acetate buffer system was added: NaAc 20mM, pH 5.0, MgCl2 10mM, DTT 2mM, NaCl 50mM. The mixture was reacted at 37°C for 1 hour to form a nucleocapsid-like structure (electron microscopy image attached). Figure 4 ); (2) Lipid delivery system for preparing NLS (nucleocapsid-like structure) vaccines Nanoparticles: a lipid delivery system was obtained by adding ((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-glycerol-3-phosphocholine (DOPC), and methoxy polyethylene glycol-N-tetradecyltetradecylamide (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 Span 85 and 0.4%-0.6% of polyoxyethylene sorbitan monooleate Tween-80 as lipid stabilizers. (3) Encapsulation: The lipid phase from step (2) and the nucleocapsid structure from step (1) are homogenized using a microfluidic device at a volume ratio of lipid:water phase = 1:3 (nitrogen-phosphorus ratio of 4~10:1) to form lipid nanoparticles with a TRR of 1:3 and a TFR of 100 ml / min.

[0022] (4) The encapsulated lipid nanoparticles were diluted 30-35 times by volume with a buffer solution containing 10 mM NaAc and 0.001% trehalose at pH 6.4, concentrated by a 100 kDa filter membrane, and then diluted again with a solution of 20 mM NaAc, 0.01% trehalose and 3.5% sucrose. After filtration and sterilization, the NLS vaccine was obtained (electron micrographs are attached). Figure 5 ).

[0023] Verification of the immunological efficacy of the obtained rabies vaccine:

[0024] The rabies virus-like nucleocapsid structure vaccine we developed in this design outperformed inactivated vaccines and mRNA vaccines that express only G proteins in terms of immunological evaluation.

[0025] Antibody detection and Elispot assay: Four groups of Blab / c mice were immunized: encapsulated NLS vaccine (20 μg mRNA + 5 μg protein), mRNA vaccine alone (containing 20 μg mRNA), conventional inactivated vaccine, and PBS blank control group. Five mice were in each group. Twenty-five mice (half male and half female) were randomly assigned to groups. Three doses were administered intramuscularly to the limbs on days 0, 7, and 21. Blood was collected from the tail vein on days 7, 21, and 35 after the first immunization. Serum was separated and RABV-G and RABV-N antibody titers were measured using ELISA. Mice were sacrificed after blood collection on day 35, and IFN-γ cytokine expression levels were detected using the Elispot assay. Results showed that the NLS vaccine produced higher levels of specific antibodies and cellular immunity than the inactivated vaccine and mRNA vaccine encoding only the G antigen. Figure 6 ).

[0026] Methods for mouse challenge protection experiment: Kunming mice were immunized with four groups of vaccines: encapsulated NLS vaccine (20 μg mRNA + 5 μg protein), mRNA vaccine alone (containing 20 μg mRNA), conventional inactivated vaccine, and PBS blank control group, with 20 mice in each group. Eighty mice (half male and half female) were randomly assigned to groups. Three doses were administered intramuscularly to the limbs on days 0, 7, and 21. The mice were challenged with 25 LD via the footpads 35 days after the first immunization. 50 The mortality rate of mice was recorded. The results showed that in the protective experiment against the virus, the vaccine achieved 100% protection in mice. Figure 7 ).

[0027] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. 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 to it in form and detail without departing from the scope defined by the claims of the present invention.

[0028] Serial number SEQ ID NO.1 Sequence name: Flavivirus 5' UTR gene fragment Length: 124 Molecular type: DNA agtaaatcct gtgtgctaat tgaggtgcat tggtctgcaa atcgagttgc taggcaataa 60 acacatttgg attaatttta atcgttcgtt gagcgattag cagagaactg accagaacgc 120 CACC Serial number SEQ ID NO.2 Sequence Name: Igκ Secretion Signal Sequence Length 60 molecular type DNA Synthetic construct of organisms gagacagaca cactcctgct atgggtactg ctgctctggg ttccaggttc cactggtgac Serial number SEQ ID NO.3 Sequence Name: Rabies Virus G Protein Sequence Length 1317 molecular type DNA agcaagggct ctaaaacctg cggattcgtc gacgagcggg gcctgtacaa gtccctgaaa 660 ggcgcctgta aactgaagct gtgcggcgtg ctgggacttc ggctcatgga cggaacatgg 720 gtcgccatcc aaacaagcaa cgagacaaag tggtgccccc ccgatcagct ggttaatctg 780 catgatttcc acagcgacga gatcgagcac ctggtggtcg aagagctggt gaaaaagcgg 840 gaagagtgcc tggatgccct ggaaagcatc atgacaacca agtccgtgag ttttcggaga 900 ctgagccacc tgaggaagct cgtgcccggc ttcggcaagg cctacaccat tttcaacaag 960 accctgatgg aagccgacgc tcactacaag tccgttagaa cctggaacga gatcatccct 1020 agcaagggat gtctgagagt gggcggcaga tgccaccccc acgtgaacgg cgtgttcttc 1080 aatggcatca tcctgggccc tgatggacac gttctgatcc cagagatgca gagctctctg 1140 ctgcagcagc acatggaact gctggaaagc agcgtgatcc cactgatgca ccctctggcc 1200 gatcctagca ccgtgtttaa ggacggcgac gaggtggagg acttcgtgga agtgcacctg 1260 cctgacgtgc ataagcaggt gtccggcgtg gacctgggcc tgcctaattg gggaaaa 1317 Serial number SEQ ID NO.4 Sequence Name: Flavivir 3'UTR Sequence Length 123 molecular type DNA gacactgttc caagcaacaa ccaagaacta gaccttgtac ataggacaaa actgaaaccg 60 ggataaaaac cacggatgga gaaccggact ccacacatca aacagaagag gatgtcagcc 120 cag Serial number SEQ ID NO.5 Sequence name polyA tail Length 138 molecular type DNA aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa gcatatgact aaaaaaaaaa 60 aaaaaaaaaa aaaaaaaaaa aaaaaaaacc gcgtgctgaa aaaaaaaaaa aaaaaaaaaa 120 aaaaaaaaaaaaaaaaaaaaaaa Serial number SEQ ID NO.6 Sequence name: Nucleic acid sequence expressing the N protein of rabies virus Length 1347 molecular type DNA agcgccattc gggtgggcac cgtggtgacc gcctacgagg actgcagcgg ccttgtgtct 720 tttaccggct tcatcaagca gatcaacctg accgcccgag aagccatcct gtacttcttc 780 cacaagaact tcgaagagga gatcagaaga atgttcgagc ctggccaaga gacagctgtg 840 ccccacagct attttatcca ctttagaagc ctgggcctga gcggaaagtc cccatactct 900 agcaatgccg tgggccacgt gttcaacctg atccacttcg tgggctgcta catgggccaa 960 gtgcggagcc tgaacgctac agtgatcgct gcctgcgccc ctcacgagat gagcgtgctg 1020 ggaggttatc tgggcgagga attcttcggc aaaggcacct tcgagagacg gtttttccgg 1080 gatgagaagg aactgcagga gtacgaggcc gctgagctga caaagtctga tgtggccctg 1140 gccgacgacg gcacagtgaa cagcgacgac gaggactact tcagcggcga aaccagatct 1200 ccagaggcag tgtacacacg gatcatgatg aacggtggaa gactgaagag aagccacatc 1260 agacggtacg tgtccgtgtc cagcaaccac caggccagac ctaattcttt cgccgagttc 1320 ctgaacaaga cctacagcaa cgacagc 1347

Claims

1. A novel rabies virus nucleocapsid nanostructure vaccine, characterized in that: The nucleocapsid nanostructure vaccine comprises an mRNA molecular structure expressing a rabies virus G protein antigen; the mRNA molecular structure expressing a rabies virus G protein antigen comprises: a gene fragment of the 5' non-coding region of a flavivirus, an Igκ secretion signal sequence, a rabies virus G protein sequence, a 3' UTR gene fragment of a 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 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 novel rabies virus nucleocapsid nanostructured vaccine is an mRNA-protein complex structure formed by combining an mRNA molecule expressing a G protein antigen with an N protein molecule.

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

4. The novel rabies virus nucleocapsid nanostructure vaccine according to claim 3, characterized in that: The method for obtaining the N protein is as follows: inserting the 6×His sequence CACCACCACCACCACCAC and the nucleic acid sequence SEQID 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 solution 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.

5. The novel rabies virus nucleocapsid nanostructure vaccine according to any one of claims 2 to 4, wherein the construction method 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 20mM, MgCl2 10mM, DTT 2mM, NaCl 50mM (all final concentrations of the system), and reacted at 37°C for 1h 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 20 mM NaAc, 0.01% trehalose and 3.5% sucrose solution. After filtration and sterilization, the NLS vaccine was obtained.

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