Self-replicating mRNA sequence, MenB prevention vaccine and preparation method

Abstract

The invention relates to the technical field of MenB vaccines, in particular to a self-replicating mRNA sequence, a MenB prevention vaccine and a preparation method of the self-replicating mRNA sequence and the MenB prevention vaccine. The self-replicating mRNA sequence comprises RNA of a coding region in an amino acid sequence, and the amino acid sequence comprises a tandem sequence formed by tandem connection of single sequences shown as SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 according to any sequence. Amino acid sequences are screened for strains with coverage found in Chinese monitoring, the vaccine for preventing MenB is provided, and the death rate caused by domestic infection due to B-type meningococcus is reduced. The vaccine for preventing MenB has the advantages of being low in injection needle number, low in dosage and high in expression effect, the dosage and frequency can be reduced, meanwhile, the expression efficiency of the vaccine is improved, and the vaccine for preventing MenB is suitable for clinical application. And a better immune effect is expected to be achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of MenB vaccines, and particularly relates to a self-replicating mRNA sequence, a preventive MenB vaccine, and a preparation method thereof. Background Art

[0002] Epidemic cerebrospinal meningitis is caused by Neisseria meningitidis. In recent years, meningitis B has been prevalent in China and many other countries, and infants and children under 5 years old are high-risk groups. Currently, the existing meningitis B vaccines do not cover the prevalent strains in China. Therefore, the development of an effective vaccine for preventing meningitis B plays an important role in its prevention and control.

[0003] The production of recombinant protein vaccines has problems such as complex processes, high costs, difficult purification, and time-consuming, which affect public acceptance. Therefore, providing an efficient and safe self-replicating mRNA molecule and a preventive MenB vaccine is the main technical problem to be solved currently. Summary of the Invention

[0004] In view of the four antigens of meningitis B, the present patent designs a self-replicating mRNA vaccine, which is encapsulated with lipid nanoparticles and encodes a tandem sequence of four antigen proteins. Through cell experiments and animal experiments, it is verified that the self-replicating mRNA vaccine molecular sequence designed by us can continuously and efficiently produce four antigens against meningitis B, and has broad application prospects.

[0005] During the experimental process, the inventors found an mRNA vaccine sequence for preventing MenB that uses a self-replicating mRNA sequence to obtain specific immune responses, high immunogenicity, long-lasting immune responses, and low vaccine costs.

[0006] The first aspect of the present invention provides a self-replicating mRNA sequence, including RNA in the coding region of the amino acid sequence, and the amino acid sequence includes a tandem sequence formed by the individual sequences shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 in any order in series.

[0007] For the strains found in the Chinese surveillance coverage, the protein sequences of the present application cover the ST-4821, ST-41 / 44, ST-32, ST-198, and ST-175 clonal complexes in Neisseria meningitidis serogroup B bacteria. The amino acid sequences are screened for Chinese strains, and the four antigen proteins of fHbp, NHBA, NadA, and PorA are optimized and the original signal peptide sequences are deleted. The optimized protein amino acid sequences include SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0008] In one embodiment, the amino acid sequence is optimized for fHbp, NHBA, NadA, and PorA antigen proteins and the original signal peptide sequence is deleted.

[0009] In one embodiment, the fHbp antigen protein molecule deletes the original 1-19 signal peptide sequence as amino acid sequence SEQ ID NO:7.

[0010] In one embodiment, the NHBA antigen protein molecule deletes the original 1-17 signal peptide sequence as amino acid sequence SEQ ID NO:8.

[0011] In one embodiment, the NadA antigen protein molecule deletes the original 1-23 signal peptide sequence as amino acid sequence SEQ ID NO:9.

[0012] In one embodiment, the PorA antigen protein molecule deletes the original 1-18 signal peptide sequence as amino acid sequence SEQ ID NO:10.

[0013] In one embodiment, one or more signal peptide sequences shown in amino acid sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6 are added at the 5'-end of the tandem sequence.

[0014] In one embodiment, the amino acid sequence is a tandem sequence formed by SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10 concatenated in any order before and after.

[0015] The inventors found during the experiment that the design based on the Chinese strain sequence can ensure that the constructed tandem sequence more precisely matches the characteristics of the MenB strains prevalent in China, thus laying the foundation for the effectiveness of the MenB vaccine. The inventors found during the experiment that different arrangements may affect the spatial structure and immunogenicity of the final protein, and the possibility of various permutations and combinations provides more options for screening the most immunologically active amino acid sequences.

[0016] In one embodiment, the signal peptide sequence added at the 5'-end of the tandem sequence includes the signal peptide of the novel coronavirus, with the sequence SEQ ID NO:1.

[0017] In one embodiment, the signal peptide sequence added at the 5'-end of the tandem sequence includes the tPA signal peptide, with the sequence SEQ ID NO:2.

[0018] In one embodiment, the signal peptide sequence added at the 5'-end of the tandem sequence includes the fHbp signal peptide, with the sequence SEQ ID NO:3.

[0019] In one embodiment, the signal peptide sequence added at the 5'-end of the tandem sequence includes the NHBA signal peptide, and the sequence is SEQ ID NO: 4.

[0020] In one embodiment, the signal peptide sequence added at the 5'-end of the tandem sequence includes the NadA signal peptide, and the sequence is SEQ ID NO: 5.

[0021] In one embodiment, the signal peptide sequence added at the 5'-end of the tandem sequence includes the PorA signal peptide, and the sequence is SEQ ID NO: 6.

[0022] In order to further optimize the tandem sequence, the inventor added a signal peptide sequence at the 5'-end of the tandem sequence to guide the nascent protein into a specific secretion pathway within the cell, ensuring that the protein can be correctly localized and function properly, and ensuring the secretion of the self-replicating enzyme and antigen; the inventor found during the experiment that different signal peptides may have different guiding characteristics and are suitable for different cell environments and protein function requirements.

[0023] In one embodiment, the signal peptide sequence shown in SEQ ID NO: 1 is added at the 5'-end of the tandem sequence.

[0024] In order to further optimize the tandem sequence, the signal peptide sequence shown in SEQ ID NO: 1 is added at the 5'-end of the tandem sequence. The inventor found during the experiment that the SEQ ID NO: 1 signal peptide performs the best in guiding the protein encoded by the tandem sequence into the secretion pathway, can significantly improve the protein expression efficiency and correct localization rate, thereby enhancing the immunogenicity of the vaccine.

[0025] In one embodiment, the flexible Linker in the amino acid sequence SEQ ID NO: 11 is used to connect between individual sequences in the tandem sequence.

[0026] In one embodiment, the flexible Linker shown in SEQ ID NO: 11 is used to connect between individual sequences in the tandem sequence.

[0027] In one embodiment, the amino acid sequence is a tandem sequence formed by SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 in any order in series. The signal peptide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 is added at the 5'-end of the tandem sequence, and the flexible Linker shown in SEQ ID NO: 11 is used to connect between individual sequences in the tandem sequence.

[0028] In one embodiment, the amino acid sequence is a tandem sequence formed by SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 concatenated in series in any order, a signal peptide sequence shown in SEQ ID NO:1 is added at the 5' end of the tandem sequence, and flexible Linker shown in SEQ ID NO:11 is used to connect between individual sequences in the tandem sequence.

[0029] During the experiment, the inventors found that in order to ensure that the protein domains encoded by individual sequences in the tandem sequence can function independently and avoid steric hindrance and interference between each other, it is defined that flexible Linker shown in SEQ ID NO:11 is used to connect between individual sequences in the tandem sequence, which can provide sufficient spatial freedom for each protein domain, enabling it to freely fold and move, thereby ensuring the correct conformation and functional integrity of the entire tandem protein.

[0030] In one embodiment, an MITD molecular sequence is added at the end of the tandem sequence, and the MITD molecular sequence includes the amino acid sequence SEQ ID NO:12 or SEQ ID NO:13.

[0031] In one embodiment, the MITD sequence is preferably SEQ ID NO:12, and the SEQ ID NO:12 sequence is more preferably the MITD sequence with a mutation design added as SEQ ID NO:13.

[0032] The inventors found in the experiment that MITD is a special sequence that can be added to vaccine antigens, which can direct the antigen to enter specific regions of dendritic cells, making the antigen more easily processed by dendritic cells and presented to T cells, thereby initiating an immune response.

[0033] For the mutation design, the inventors found in the experiment the wild-type sequence SEQ ID NO:27 based on the antigen fHbp and the mutated sequence SEQ ID NO:28 based on the antigen fHbp.

[0034] For the mutation design, the inventors found in the experiment the wild-type sequence SEQ ID NO:29 based on the antigen NHBA and the mutated sequence SEQ ID NO:30 based on the antigen NHBA.

[0035] For the mutation design, the inventors found in the experiment the wild-type sequence SEQ ID NO:31 based on the antigen NadA and the mutated sequence SEQ ID NO:32 based on the antigen NadA.

[0036] For the mutant design inventors, in the experiment, the sequence SEQ ID NO: 33 based on the wild-type of antigen PorA and the mutated sequence SEQ ID NO: 34 based on antigen PorA were discovered.

[0037] In one embodiment, the self-replicating mRNA sequence includes target protein amino acids designed by molecular tandem for fHbp, NHBA, NadA, and PorA antigen proteins.

[0038] In one embodiment, the self-replicating mRNA sequence includes RNA of the coding region of the amino acid sequence provided by the first group of experiments.

[0039] In one embodiment, molecular tandem design is performed on four antigen proteins, fHbp, NHBA, NadA, and PorA, as the self-replicating mRNA sequence.

[0040] In one embodiment, molecular tandem design is performed on four antigen proteins, fHbp, NHBA, NadA, and PorA, to obtain the target protein amino acids of novel coronavirus SP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA, and the target protein amino acid sequence is SEQ ID NO: 14.

[0041] In one embodiment, by adjusting the codon sequence of the target protein amino acid sequence SEQ ID NO: 14 and optimizing its GC content and secondary structure, the optimized base sequence is SEQ ID NO: 15.

[0042] In one embodiment, molecular tandem design is performed on four antigen proteins, fHbp, NHBA, NadA, and PorA, to obtain the target protein amino acids of tPASP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA, and the target protein amino acid sequence is SEQ ID NO: 16.

[0043] In one embodiment, by adjusting the codon sequence of the target protein amino acid sequence SEQ ID NO: 16 and optimizing its GC content and secondary structure, the optimized base sequence is SEQ ID NO: 17.

[0044] In one embodiment, molecular tandem design is performed on four antigen proteins, fHbp, NHBA, NadA, and PorA, to obtain the target protein amino acids of fHbpSP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA, and the target protein amino acid sequence is SEQ ID NO: 18.

[0045] In one embodiment, the codon sequence of the target protein amino acid sequence SEQ ID NO: 18 was adjusted to optimize its GC content and secondary structure, and the optimized base sequence was SEQ ID NO: 19.

[0046] In one embodiment, molecular tandem design was performed on four antigen proteins, fHbp, NHBA, NadA, and PorA, to obtain the target protein amino acid of NHBASP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA, and the target protein amino acid sequence was SEQ ID NO: 20.

[0047] In one embodiment, the codon sequence of the target protein amino acid sequence SEQ ID NO: 20 was adjusted to optimize its GC content and secondary structure, and the optimized base sequence was SEQ ID NO: 21.

[0048] In one embodiment, molecular tandem design was performed on four antigen proteins, fHbp, NHBA, NadA, and PorA, to obtain the target protein amino acid of NadASP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA, and the target protein amino acid sequence was SEQ ID NO: 22.

[0049] In one embodiment, the codon sequence of the target protein amino acid sequence SEQ ID NO: 22 was adjusted to optimize its GC content and secondary structure, and the optimized base sequence was SEQ ID NO: 23.

[0050] In one embodiment, molecular tandem design was performed on four antigen proteins, fHbp, NHBA, NadA, and PorA, to obtain the target protein amino acid of PorASP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA, and the target protein amino acid sequence was SEQ ID NO: 24.

[0051] In one embodiment, the codon sequence of the target protein amino acid sequence SEQ ID NO: 24 was adjusted to optimize its GC content and secondary structure, and the optimized base sequence was SEQ ID NO: 25.

[0052] In one embodiment, the sequence termination codon of the self-replicating mRNA includes the amino acid sequence SEQ ID NO: 26.

[0053] During the experiment, the inventors found that to achieve precise control of protein translation, protein synthesis can be terminated by adding a stop codon, thereby producing the desired protein variants or avoiding the production of unnecessary proteins. In particular, the stop codon sequence is designed as SEQ ID NO: 26.

[0054] During the experiment, the inventors found that to improve gene expression levels, according to the codon usage preference of the host cell, high-frequency synonymous codons are selected; the stability of the secondary structure of the mRNA sequence is closely related to the half-life of the mRNA sequence and the protein expression level. Enhancing the stability of the secondary structure of the mRNA sequence is beneficial to the expression of the target protein; a high content of guanine and cytosine (G and C) can improve the stability and translation efficiency of the mRNA sequence. By adjusting the GC content in the codon sequence of the target gene, the expression level in the host cell can be improved. By adjusting the GC content of the codon sequence of the target gene, the GC contents in the optimized amino acid sequences SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25 are 58.85%, 58.34%, 58.58%, 58.10%, 58.88%, and 58.77% respectively.

[0055] In one embodiment, the self-replicating mRNA sequence comprises one or more sequences shown in SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 25.

[0056] In one embodiment, the self-replicating mRNA sequence is the sequence shown in SEQ ID NO: 15.

[0057] In one embodiment, the self-replicating mRNA sequence is the sequence shown in SEQ ID NO: 17.

[0058] In one embodiment, the self-replicating mRNA sequence is the sequence shown in SEQ ID NO: 19.

[0059] In one embodiment, the self-replicating mRNA sequence is the sequence shown in SEQ ID NO: 21.

[0060] In one embodiment, the self-replicating mRNA sequence is the sequence shown in SEQ ID NO: 23.

[0061] In one embodiment, the self-replicating mRNA sequence is the sequence shown in SEQ ID NO: 25.

[0062] In one embodiment, the self-replicating mRNA sequence comprises the amino acid sequence shown in SEQ ID NO: 17.

[0063] The second aspect of the present invention provides a prophylactic MenB vaccine comprising a self-replicating mRNA sequence.

[0064] The third aspect of the present invention provides a method for preparing a prophylactic MenB vaccine, comprising the following steps: Vector construction, plasmid preparation, preparation of the mRNA stock solution, and encapsulation of the mRNA stock solution to obtain the prophylactic MenB vaccine.

[0065] In one embodiment, the method for preparing the prophylactic MenB vaccine comprises the following steps: S1. Vector construction: The self-replicating mRNA sequence is cloned into a self-replicating vector by double digestion with SalI / XbaI to construct a self-replicating mRNA plasmid; then, the DNA sequence of the self-replicating mRNA plasmid is introduced into Escherichia coli cells by heat shock to establish a seed bank; S2. Plasmid preparation: ① Fermentation culture: Take the engineering bacteria seeds containing the plasmid from the seed bank, inoculate them into an antibiotic screening medium, and perform large-scale culture in a fermenter to allow the plasmid to replicate in large quantities to obtain a culture; ② Crude extraction: The culture in the fermenter is centrifuged to obtain bacterial sludge, and a resuspension solution is successively added to the bacterial sludge for mixing, standing, removing floating impurities, filtering, and ultrafiltration concentration to obtain crude plasmid DNA; ③ Purification: Purify the crude plasmid DNA to obtain supercoiled plasmid; ④ Linearization: Cut the supercoiled plasmid with a restriction endonuclease, and purify it by anion chromatography to obtain a linearized plasmid template; S3. Preparation of the mRNA stock solution: ① Synthesize mRNA, mix the linearized plasmid DNA template, RNA polymerase, and NTPs to synthesize mRNA; ② Purification, purify the synthesized mRNA to obtain the mRNA stock solution; S4. Encapsulation of the mRNA stock solution: ① Obtain nanoparticles, dilute the mRNA stock solution as the aqueous phase, and mix the aqueous phase with the organic phase and prepare nanoparticles through a microfluidic control instrument; ② Solidification and purification, add PBS buffer to the nanoparticles for solidification to obtain LNP particles, and then perform purification and filtration to obtain the LNP-encapsulated drug composition of mRNA, namely the prophylactic MenB vaccine.

[0066] In one embodiment, the method for preparing the prophylactic MenB vaccine comprises the following steps: S1. Construction of recombinant strain: Ⅰ. Construction of recombinant plasmid: ① According to the optimized self-replicating mRNA sequences in Examples 1, 2, 3, 4, 5, and 6 of the second group of experiments, after sequencing verification, the 4-antigen ligation gene with SalI carried upstream and XbaI carried downstream and the self-replicating vector JJ-Sap-2 were double digested with SalI+XbaI. ② Recover the digested 4-antigen ligation gene and self-replicating vector, prepare a reaction system according to the molar ratio of the ligation gene:vector of 3-5:1, add T4 DNA ligase and reaction buffer, mix evenly and connect at room temperature for 30-60 min or connect overnight at 16°C to obtain the recombinant plasmid.

[0067] Ⅱ. Obtaining of recombinant strain: ① Take 3-5 μL of the ligation product of the recombinant plasmid and add it to 50 μL of thawed competent cells DH5α, mix well and perform heat shock at a heat shock temperature of 42°C for 30-60 s; ② Add the heat-shocked Escherichia coli cells to 500 μL of liquid LB medium, culture in a constant temperature shaking incubator at 37°C and 200 rpm for about one hour, then spread on solid LB medium and culture in a constant temperature incubator at 37°C for 16-18 hours.

[0068] S2. Preparation of recombinant plasmid: ① Select well-growing single colonies and add them to liquid synthetic medium, culture in a constant temperature shaking incubator at 37°C and 200 rpm for 4-6 hours, take a small amount of bacterial liquid, extract the plasmid with a plasmid extraction kit and then perform SalI+XbaI digestion identification; ② Expand the culture of the bacterial liquid with correct digestion identification results in a constant temperature shaking incubator at 37°C and 200 rpm for 14-16 hours, centrifuge at 4000 rpm for about 5-10 min to harvest the bacterial pellet; ③ Lyse the collected bacterial pellet, add Lysis Solution I, use a shaker to fully suspend the bacteria, and the volume ratio of the bacterial pellet to Lysis Solution I is 4-6:250; after the bacteria are fully suspended, add Lysis Solution II, gently invert and mix to lyse the bacteria; add Lysis Solution III, immediately gently invert and mix to fully neutralize it, and let it stand at room temperature for 5-10 min. The volume ratio of Lysis Solution I:Lysis Solution II:Lysis Solution III is 5:5:7; centrifuge the lysed mixture at 12000 rpm for 5-10 min, take the supernatant and add it to the DNA adsorption column, centrifuge at 12000 rpm for 1 min to remove the waste liquid; add the washing solution to the adsorption column, centrifuge at 12000 rpm for 1 min to remove the waste liquid; add the elution solution, let it stand at 37°C for 2-5 min and then centrifuge at 12000 rpm for 1 min, collect the elution solution to obtain the DNA solution of the circular plasmid.

[0069] Preparation of S3.mRNA stock solution: Ⅰ. Plasmid linearization: ① Use the BspQ1 digestion system, incubate at 50 °C for 1 - 2 hours to digest the circular plasmid into a linearized plasmid, and take a part of the digestion product for verification by agarose gel electrophoresis; ② Add 0.5 times the volume of magnetic beads to the remaining digestion product and mix well. Incubate at room temperature for 10 - 15 min and then place on a magnetic stand. After the solution becomes clear, remove the supernatant; ③ Wash the magnetic beads with freshly prepared 80% ethanol solution, remove the supernatant and dry the magnetic beads. Add an appropriate volume of Nuclease - free H2O, pipette and mix well, then aspirate the supernatant to obtain a purified linear plasmid DNA solution, and store it at -20 °C.

[0070] Ⅱ. In Vitro Transcription Reaction IVT (In Vitro Transcription) ① Mix the linear plasmid DNA template, NTPs, CleanCap AU, buffer and other substrates, add T7 RNA polymerase, mix well, and place in a 37 °C PCR instrument for in vitro transcription reaction. After reacting for 1 - 3 hours, add DNase I to terminate the reaction; ② Add Nuclease - free water and LiCl solution to the reacted solution. The volume ratio of the reacted solution, water and LiCl is 1:1.5:1.5. Invert and mix well, then incubate at -20 °C for 15 - 30 min, centrifuge at 4 °C, 12000g for 10 - 15 min to remove the waste liquid, add 70% ethanol to wash the impurities, remove the supernatant, and add Nuclease - free water to dissolve the precipitate to obtain the mRNA stock solution, and store it at -80 °C.

[0071] S4. Encapsulation of mRNA stock solution ① Thaw the mRNA stock solution in a cold water bath, and dilute the mRNA stock solution with citrate buffer to a concentration of about 0.1 - 0.5 mg / ml to obtain the mRNA working solution; ② Encapsulate the self - replicating mRNA in LNP. The molar concentration ratio of cations, DSPC, CHO - HP and DMP - PEG2000 in the lipid phase is (30 - 60):(5 - 10):(30 - 60):(2 - 5), and the total molar concentration of the lipid phase is 15 - 20 mmol / L; the cations in the lipid phase include DLin - MC3 - DMA, JK - 0042, JK - 0043, JK - 0045.

[0072] ③ The volume ratio of the organic phase to the mRNA working solution is 1:(2 - 4), and nanoparticles LNP are prepared by a microfluidic control instrument; ④ Add PBS buffer in a volume 5 - 10 times that of the harvested nanoparticles for dilution, and concentrate by centrifugation using a 100KD ultrafiltration centrifugal tube at 4°C and 1500g. When the volume is concentrated to approximately the volume of the initially obtained LNP, add PBS buffer in a volume 5 - 10 times again for dilution, and continue to concentrate to the expected concentration volume. After filtering through a 0.2μm membrane, the finished LNP product is obtained, and the MenB preventive vaccine is obtained.

[0073] The MenB preventive vaccine is stored at -20°C.

[0074] In one embodiment, the preparation method of the MenB preventive vaccine includes the following steps: S1. Vector construction: Clone the self-replicating mRNA sequence into the self-replicating vector by double digestion with SalI / XbaI to construct a self-replicating mRNA plasmid; then introduce the DNA sequence of the self-replicating mRNA plasmid into Escherichia coli cells by heat shock method to establish a research and development seed bank; S2. Plasmid preparation: ① Take the engineering bacteria seeds from the research and development seed bank and add them to a liquid synthetic medium in a shaking incubator, and detect that the OD600 of the seeds reaches 0.6 - 2.5. Amplify the cultured seeds and transfer them to a fermenter for large-scale culture for 38 - 45 hours. The fermentation temperature is controlled at 28°C - 37°C, the rotation speed is 100rpm - 1000rpm, the dissolved oxygen is related to the stirring speed, the pH is controlled at 6.3 - 7.0, and the feeding is related to the pH; after the fermentation is completed, centrifuge at 4000rpm for about 5 minutes to remove the supernatant and harvest the bacterial sludge; ② Mix the bacterial sludge with the resuspension S1 at a mass ratio of 1:5 - 15. Add the S2 solution in a proportion 1 - 3 times that of the S1 solution, let it stand for 2 - 10 minutes, then add 1 - 5 times the S3 solution and ammonium bicarbonate, and let it stand for 30 - 60 minutes; remove the floating impurities and filter using a filter; concentrate the solution using an ultrafiltration system; the pH value of the S1 solution is 7.5 - 8.5, and it is composed of 25mM - 100mM glucose, 15mM - 35mM Tri-HCl, and 5mM - 20mM EDTA; the S2 solution is composed of a freshly prepared solution of 0.1M - 0.3M NaOH and 0.5% - 2% SDS; the S3 solution is composed of a mixed solution formed by 3M KAc and 2M HAc.

[0075] ③ Perform chromatography using a molecular sieve chromatography column. The linear flow rate of chromatography is 1 - 4 cm / h. At about 1 / 3CV after the sample loading starts, the target product will flow through and the UV curve will rise. Collect this flow-through peak. The buffer solution used for chromatography with the molecular sieve chromatography column is composed of 1.8M - 2.3M (NH4)2SO4, 0.2M - 0.5M NaCl, 0.1M Tris-HCl, and 0.01M EDTA-2Na.

[0076] ④Perform chromatography using an affinity chromatography column, equilibrate with buffer solution A, and the linear flow rate of chromatography is 10 - 15 cm / h. After sample loading, wash impurities with 5% - 15% buffer solution B for about 2 - 5 CV.

[0077] The buffer solution A consists of 1.8 M - 2.3 M (NH4)2SO4, 0.1 M Tris - HCl, and 0.01 M EDTA - 2Na; The buffer solution B consists of 1.8 M - 2.3 M (NH4)2SO4, 0.2 M - 0.5 M NaCl, 0.1 M Tris - HCl, and 0.01 M EDTA - 2Na; and elute with 20% - 50% buffer solution B, and collect the elution peak.

[0078] ⑤Use an ultrafiltration system to change the solution, change the salt solution to 1×TE buffer solution, and select a hollow fiber column with a molecular weight cut - off of 100 kD - 500 kD.

[0079] ⑥Mix supercoiled plasmid DNA, linearizing enzyme, and water, incubate at 45℃ - 55℃ for 0.5 - 4 hours, and purify using anion chromatography column chromatography and ultrafiltration to obtain a linearized plasmid DNA template, which is stored at - 80℃; the linear flow rate of chromatography is 10 - 15 cm / h, the chromatography solution A consists of EDTA - 2Na and Tris - HCl, the mass ratio of EDTA - 2Na to Tris - HCl is 1.681:7.88, and the pH of chromatography solution A is 7.5; The chromatography solution B consists of EDTA - 2Na and Tris - HCl, the mass ratio of EDTA - 2Na to Tris - HCl is 1.681:7.88, and the pH of chromatography solution B is 7.5.

[0080] Preparation of the mRNA stock solution: ①Mix the linearized plasmid DNA template, NTPs, and buffer substrate, then add polymerase, and react at 33℃ - 37℃ in a synthesis workstation for 1 - 3 hours; ②Purify by affinity chromatography column chromatography, and select OligodT as the chromatography packing; dilute the reaction solution 15 - 30 times with the equilibration solution before purification, the linear flow rate of purification is 6 cm / h - 20 cm / h, and wash impurities for 2 - 8 CV. The equilibration solution consists of NaCl, EDTA, and TrisHCl, and their mass ratio is 9.35:0.067:0.32; after washing impurities, elute with pure water and collect the elution peak; ③Purify using a TFF system, and the replacement solution is pure water. The transmembrane pressure is 5 - 20 psi, the shear rate is 1000 - 4000 / sec, wash and filter 8 - 15 times and then concentrate to the target concentration. Filter the obtained sample with 0.2 μm to obtain the mRNA stock solution, which is stored at - 80℃.

[0081] Encapsulation of S4.mRNA stock solution: ① Thaw the mRNA stock solution in a cold water bath, and dilute the mRNA stock solution with buffer salts to a concentration of 0.1 mg / ml - 0.5 mg / ml, and label it as RNA working solution.

[0082] The organic phase: The volume ratio to the aqueous phase RNA working solution is 1:3, and nanoparticles are prepared by a microfluidic control preparation instrument.

[0083] ② Add 1×PBS with a volume 4 - 20 times that of the harvested nanoparticles to solidify the LNP particles, and then use a TFF ultrafiltration system for purification. The replacement solution is 1×PBS. The transmembrane pressure is 10 psi, the shear rate is 2000 / sec, dilute and concentrate 4 - 8 times, wash and filter 4 - 8 cycles, and then concentrate 2 - 3 times. The obtained sample is filtered through 0.2 μm to obtain the LNP-encapsulated drug composition of mRNA, and the preventive MenB vaccine is obtained and stored at -20°C.

[0084] During the experiment, the inventors found that the preventive MenB vaccine obtained by using the above preparation method of the preventive MenB vaccine showed the effects of low injection times, low dose, and high expression. The possible reason is that saRNA can self-replicate in cells as a vector, which can reduce the dosage and frequency of administration, and at the same time improve the expression efficiency of the vaccine, in order to achieve better immune effects.

[0085] During the experiment, the inventors found that the preventive MenB vaccines in different formulation forms obtained by using the above preparation method of the preventive MenB vaccine can improve the efficacy of the vaccine, and it can be shown in the efficacy experiment that the preventive MenB vaccines in different formulation forms will have different effects on the efficacy. The vaccine after process improvement has the advantages of quickly triggering an immune response, high antibody level, and providing long-term immune protection. Description of the Drawings

[0086] Figure 1 It is the electrophoresis test result obtained for the self-replicating mRNA sequence SEQ ID NO:15 and its recombinant plasmid.

[0087] Figure 2 It is the electrophoresis test result obtained for the self-replicating mRNA sequence SEQ ID NO:17 and its recombinant plasmid.

[0088] Figure 3 It is the electrophoresis test result obtained for the self-replicating mRNA sequence SEQ ID NO:19 and its recombinant plasmid.

[0089] Figure 4 It is the electrophoresis test result obtained for the self-replicating mRNA sequence SEQ ID NO:21 and its recombinant plasmid.

[0090] Figure 5 Electrophoresis test results obtained for the self-replicating mRNA sequence SEQ ID NO: 23 and its recombinant plasmid.

[0091] Figure 6 Electrophoresis test results obtained for the self-replicating mRNA sequence SEQ ID NO: 25 and its recombinant plasmid.

[0092] Figure 7 Results of protein band detection obtained by immunoblotting for the sequences provided in Example 7, Example 8, Example 9, Example 10, Example 11, and Example 12.

[0093] Figure 8 Graph of fHbp binding antibody detection results.

[0094] Figure 9 Graph of NHBA binding antibody detection results.

[0095] Figure 10 Graph of NadA binding antibody detection results.

[0096] Figure 11 Graph of porA binding antibody detection results.

[0097] Figure 12 Graph of FACs detection results on day D34 after injection of the MenB preventive vaccine.

[0098] Figure 13 Graph of FACs detection results on day D70 after injection of the MenB preventive vaccine.

[0099] Figure 14 Graph of ELISPOT detection results on day D34 after injection of the MenB preventive vaccine.

[0100] Figure 15 Graph of ELISPOT detection results on day D70 after injection of the MenB preventive vaccine.

[0101] Figure 16 Results of MenB (PorA) binding antibody titer detection.

[0102] Figure 17 Results of MenB (NadA) binding antibody titer detection.

[0103] Figure 18 Results of MenB (NHBA) binding antibody titer detection.

[0104] Figure 19 Results of MenB (fHbp) binding antibody titer detection.

[0105] Figure 20 Results of CD4+IFNg+ T cell typing detection

[0106] Figure 21 Results of CD4+CD69+ T cell typing detection

[0107] Figure 22 Results of CD8+IFNg+ T cell typing detection

[0108] Figure 23 Results of CD8+CD69+ T cell typing detection

[0109] Figure 24 Results of ELISPOT detection on day 35 after injection of D

[0110] Figure 25 Results of ELISPOT detection on day 70 after injection of D

[0111] Figure 26 Results of serum bactericidal titer detection of SEQ ID NO:17 split - formulation mixture

[0112] Figure 27 Results of serum bactericidal titer detection of SEQ ID NO:15 split - formulation mixture

[0113] Figure 16 In a, when the dosage of the self - replicating mRNA sequence SEQ ID NO:15 provided in Example 1, the self - replicating mRNA sequence SEQ ID NO:17 provided in Example 2, the SEQ ID NO:17 - split - stock solution mixture provided in Example 15, and the split - formulation mixture provided in Example 16 is 1 μg, the results of serum binding antibody detection by binding to PorA antigen protein

[0114] Figure 16 In b, when the dosage of the self - replicating mRNA sequence SEQ ID NO:15 provided in Example 1, the self - replicating mRNA sequence SEQ ID NO:17 provided in Example 2, the SEQ ID NO:17 - split - stock solution mixture provided in Example 15, and the split - formulation mixture provided in Example 16 is 5 μg, the results of serum binding antibody detection by binding to PorA antigen protein

[0115] Figure 16 In c, when the dosage of the self - replicating mRNA sequence SEQ ID NO:15 provided in Example 1, the self - replicating mRNA sequence SEQ ID NO:17 provided in Example 2, the SEQ ID NO:17 - split - stock solution mixture provided in Example 15, and the split - formulation mixture provided in Example 16 is 15 μg, the results of serum binding antibody detection by binding to PorA antigen protein

[0116] Figure 17 In a, the results of serum binding antibody detection by binding to NadA antigen protein when the dosage of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17-split-stock solution mixture provided in Example 15, and the split-formulation mixture provided in Example 16 is 1 μg are shown.

[0117] Figure 17 In b, the results of serum binding antibody detection by binding to NadA antigen protein when the dosage of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17-split-stock solution mixture provided in Example 15, and the split-formulation mixture provided in Example 16 is 5 μg are shown.

[0118] Figure 17 In c, the results of serum binding antibody detection by binding to NadA antigen protein when the dosage of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17-split-stock solution mixture provided in Example 15, and the split-formulation mixture provided in Example 16 is 15 μg are shown.

[0119] Figure 18 In a, the results of serum binding antibody detection by binding to NHBA antigen protein when the dosage of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17-split-stock solution mixture provided in Example 15, and the split-formulation mixture provided in Example 16 is 1 μg are shown.

[0120] Figure 18 In b, the results of serum binding antibody detection by binding to NHBA antigen protein when the dosage of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17-split-stock solution mixture provided in Example 15, and the split-formulation mixture provided in Example 16 is 5 μg are shown.

[0121] Figure 18Among them, c is the result of serum binding antibody detection by binding to NHBA antigen protein when the dosage of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17-split-stock solution mixture provided in Example 15, and the split-formulation mixture provided in Example 16 is 15 μg.

[0122] Figure 19 Among them, a is the result of serum binding antibody detection by binding to fHbp antigen protein when the dosage of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17-split-stock solution mixture provided in Example 15, and the split-formulation mixture provided in Example 16 is 1 μg.

[0123] Figure 19 Among them, b is the result of serum binding antibody detection by binding to fHbp antigen protein when the dosage of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17-split-stock solution mixture provided in Example 15, and the split-formulation mixture provided in Example 16 is 5 μg.

[0124] Figure 19 Among them, c is the result of serum binding antibody detection by binding to fHbp antigen protein when the dosage of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17-split-stock solution mixture provided in Example 15, and the split-formulation mixture provided in Example 16 is 15 μg.

[0125] Figure 24 Among them, a is the result of ELISPOT detection on day D35 after injecting into mice when the dosage of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15 split-formulation mixture provided in Example 14 is 1 μg.

[0126] Figure 24In this, b is the result of ELISPOT assay on day D35 after injecting into mice at a dose of 5 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15 split-formulation mixture provided in Example 14.

[0127] Figure 24 In this, c is the result of ELISPOT assay on day D35 after injecting into mice at a dose of 5 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15 split-formulation mixture provided in Example 14.

[0128] Figure 25 In this, a is the result of ELISPOT assay on day D70 after injecting into mice at a dose of 1 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15 split-formulation mixture provided in Example 14.

[0129] Figure 25 In this, b is the result of ELISPOT assay on day D70 after injecting into mice at a dose of 5 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15 split-formulation mixture provided in Example 14.

[0130] Figure 25 In this, c is the result of ELISPOT assay on day D70 after injecting into mice at a dose of 5 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15 split-formulation mixture provided in Example 14.

[0131] Beneficial effects 1. This application screens amino acid sequences for strains found in Chinese surveillance for coverage, provides a preventive MenB vaccine, and reduces the mortality rate of domestic infections caused by Neisseria meningitidis serogroup B.

[0132] 2. In this application, for the amino acids that can mutate, the MITD molecular sequence is added to the end of the tandem sequence, which can direct the antigen to a specific region of dendritic cells, making the antigen more easily processed by dendritic cells and presented to T cells, thereby initiating an immune response.

[0133] 3. In this application, a signal peptide sequence is added at the 5'-end of the tandem sequence to guide the newly synthesized protein into a specific secretory pathway within the cell, ensuring that the protein can be correctly localized and function properly, and ensuring the secretion of the self-replicating enzyme and the antigen; the inventor found during the experiment that different signal peptides may have different guiding characteristics and are suitable for different cell environments and protein function requirements.

[0134] 4. To achieve precise control of protein translation in this application, the synthesis of the optimized protein can be terminated by adding a stop codon, thereby generating the desired protein variant or avoiding the production of unnecessary proteins.

[0135] 5. The tandem sequence provided in this application can ensure that the protein domains encoded by each individual sequence in the tandem sequence can function independently, avoiding steric hindrance and interference between each other. It is specified that a flexible Linker shown in SEQ ID NO: 11 is used between individual sequences of the tandem sequence, which can provide sufficient spatial freedom for each protein domain, enabling it to freely fold and move, thereby ensuring the correct conformation and functional integrity of the entire tandem protein.

[0136] 6. In this application, saRNA can self-replicate in cells as a vector, enabling the MenB vaccine to exhibit low injection doses, low dosages, and high expression effects, reducing the dosage and frequency of administration, and simultaneously improving the expression efficiency of the vaccine, in order to achieve a better immune effect. Detailed implementation manners

[0137] To better understand the present invention, the following describes the embodiments in detail with reference to the accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and cannot limit the scope of the present invention.

[0138] Unless otherwise specified, the technical means adopted in these embodiments are conventional technical means in the art.

[0139] The first group of experiments The first group of experiments screened amino acid sequences.

[0140] For the strains found in the coverage monitoring in China, the protein sequences of the present application cover the ST-4821, ST-41 / 44, ST-32, ST-198, and ST-175 clonal complexes in Neisseria meningitidis serogroup B bacteria. Amino acid sequences are screened for Chinese strains, and the four antigen proteins of fHbp, NHBA, NadA, and PorA are optimized and the original signal peptide sequences are deleted. The amino acid sequences are SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10.

[0141] The fHbp antigen protein molecule deletes the original 1-19 signal peptide sequence as the amino acid sequence SEQ ID NO:7.

[0142] The NHBA antigen protein molecule deletes the original 1-17 signal peptide sequence as the amino acid sequence SEQ ID NO:8.

[0143] The NadA antigen protein molecule deletes the original 1-23 signal peptide sequence as the amino acid sequence SEQ ID NO:9.

[0144] The PorA antigen protein molecule deletes the original 1-18 signal peptide sequence as the amino acid sequence SEQ ID NO:10.

[0145] The second group of experiments The second group of experiments provides self-replicating mRNA sequences, and molecular tandem designs are performed on the four antigen proteins of fHbp, NHBA, NadA, and PorA as self-replicating mRNA sequences.

[0146] The second group of experiments includes Example 1, Example 2, Example 3, Example 4, Example 5, and Example 6.

[0147] The self-replicating mRNA sequence provided in Example 1 is SEQ ID NO:15.

[0148] The self-replicating mRNA sequence provided in Example 2 is SEQ ID NO:17.

[0149] The self-replicating mRNA sequence provided in Example 3 is SEQ ID NO:19.

[0150] The self-replicating mRNA sequence provided in Example 4 is SEQ ID NO:21.

[0151] The self-replicating mRNA sequence provided in Example 5 is SEQ ID NO:23.

[0152] The self-replicating mRNA sequence provided in Example 6 is SEQ ID NO:25.

[0153] The self-replicating mRNA sequence includes the RNA of the coding region in the amino acid sequences provided by the first set of experiments, and the amino acid sequences are formed into a tandem sequence by concatenating the individual sequences shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10 in any order before and after.

[0154] The signal peptide sequence added at the 5' end of the tandem sequence is the signal peptide of the novel coronavirus, and the sequence is SEQ ID NO:1.

[0155] The tandem sequence is connected between individual sequences using the flexible Linker shown in SEQ ID NO:11.

[0156] The mutant-designed MITD molecular sequence is added to the end of the tandem sequence, and the MITD molecular sequence is the amino acid sequence SEQ ID NO:13.

[0157] Example 1 Based on the self-replicating mRNA sequence provided by the second set of experiments, in this example, molecular tandem design is carried out for four antigen proteins, fHbp, NHBA, NadA, and PorA, to obtain the amino acids of the target protein of the novel coronavirus SP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA, and the amino acid sequence is SEQ ID NO:14.

[0158] In this example, by adjusting the codon sequence of the target gene SEQ ID NO:14, optimizing its GC content and secondary structure, the optimized base sequence SEQ ID NO:15 is obtained, and the self-replicating mRNA sequence is obtained.

[0159] Example 2 Based on the self-replicating mRNA sequence provided by the second set of experiments, in this example, molecular tandem design is carried out for four antigen proteins, fHbp, NHBA, NadA, and PorA, to obtain the amino acids of the target protein tPASP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA, and the amino acid sequence of the target protein is SEQ ID NO:16.

[0160] In this example, by adjusting the codon sequence of the target gene SEQ ID NO:16, optimizing its GC content and secondary structure, the optimized base sequence SEQ ID NO:17 is obtained, and the self-replicating mRNA sequence is obtained.

[0161] Example 3 Based on the self-replicating mRNA sequences provided by the second group of experiments, in this example, molecular tandem design was carried out for four antigen proteins, namely fHbp, NHBA, NadA, and PorA, to obtain the target protein amino acids of fHbpSP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA. The amino acid sequence of the target protein is SEQ ID NO: 18.

[0162] In this example, by adjusting the codon sequence of the target gene SEQ ID NO: 18, optimizing its GC content and secondary structure, the optimized base sequence SEQ ID NO: 19 was obtained, and the self-replicating mRNA sequence was obtained.

[0163] Example 4 Based on the self-replicating mRNA sequences provided by the second group of experiments, in this example, molecular tandem design was carried out for four antigen proteins, namely fHbp, NHBA, NadA, and PorA, to obtain the target protein amino acids of NHBASP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA. The amino acid sequence of the target protein is SEQ ID NO: 20.

[0164] In this example, by adjusting the codon sequence of the target gene SEQ ID NO: 20, optimizing its GC content and secondary structure, the optimized base sequence SEQ ID NO: 21 was obtained, and the self-replicating mRNA sequence was obtained.

[0165] Example 5 Based on the self-replicating mRNA sequences provided by the second group of experiments, in this example, molecular tandem design was carried out for four antigen proteins, namely fHbp, NHBA, NadA, and PorA, to obtain the target protein amino acids of NadASP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA. The amino acid sequence of the target protein is SEQ ID NO: 22.

[0166] In this example, by adjusting the codon sequence of the target gene SEQ ID NO: 22, optimizing its GC content and secondary structure, the optimized base sequence SEQ ID NO: 23 was obtained, and the self-replicating mRNA sequence was obtained.

[0167] Example 6 Based on the self-replicating mRNA sequences provided by the second group of experiments, in this example, molecular tandem design was carried out for four antigen proteins, namely fHbp, NHBA, NadA, and PorA, to obtain the target protein amino acids of PorASP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA. The amino acid sequence of the target protein is SEQ ID NO: 24.

[0168] In this embodiment, the codon sequence of the target gene SEQ ID NO: 24 was adjusted to optimize its GC content and secondary structure, resulting in the optimized base sequence SEQ ID NO: 25, and a self-replicating mRNA sequence was obtained.

[0169] The second group of experiments provided a self-replicating mRNA sequence, and the self-replicating mRNA sequence was optimized using SEQ ID NO: 26 as the stop codon.

[0170] The self-replicating mRNA sequence includes the amino acid sequence shown in SEQ ID NO: 17.

[0171] The third group of experiments The third group of experiments provided a preventive MenB vaccine and a preparation method thereof.

[0172] The preventive MenB vaccine includes the self-replicating mRNA sequence provided by the second group of experiments.

[0173] The third group of experiments includes Example 7, Example 8, Example 9, Example 10, Example 11, and Example 12.

[0174] The self-replicating mRNA sequence used in the preventive MenB vaccine provided by Example 7 is SEQ ID NO: 15.

[0175] The self-replicating mRNA sequence used in the preventive MenB vaccine provided by Example 8 is SEQ ID NO: 17.

[0176] The self-replicating mRNA sequence used in the preventive MenB vaccine provided by Example 9 is SEQ ID NO: 19.

[0177] The self-replicating mRNA sequence used in the preventive MenB vaccine provided by Example 10 is SEQ ID NO: 21.

[0178] The self-replicating mRNA sequence used in the preventive MenB vaccine provided by Example 11 is SEQ ID NO: 23.

[0179] The self-replicating mRNA sequence used in the preventive MenB vaccine provided by Example 12 is SEQ ID NO: 25.

[0180] The preparation methods of the preventive MenB vaccines provided by Example 7, Example 8, Example 9, Example 10, Example 11, and Example 12 were prepared according to the following method steps.

[0181] The preparation method of the preventive MenB vaccine includes the following steps: S1. Construction of recombinant strains: Ⅰ. Construction of recombinant plasmids: ① According to the self-replicating mRNA sequences optimized in Examples 1, 2, 3, 4, 5, and 6 of the second group of experiments, after the sequencing verification was correct, the 4-antigen ligation gene carrying SalI upstream and XbaI downstream and the self-replicating vector JJ-Sap-2 were double digested with SalI+XbaI; ② Recover the digested 4-antigen ligation gene and self-replicating vector, prepare the reaction system according to the molar ratio of the ligation gene to the vector of 5:1, add T4 DNA ligase and reaction buffer, mix evenly and connect at room temperature for 30 min to obtain the recombinant plasmid.

[0182] II. Obtaining recombinant strains: ① Take 5 μL of the ligation product of the recombinant plasmid and add it to 50 μL of the thawed competent cell DH5α, mix well and perform heat shock, the heat shock temperature is 42 °C, and the heat shock time is 60 s; ② Add the heat-shocked Escherichia coli cells to 500 μL of liquid LB medium, culture them in a constant temperature shaking incubator at 37 °C and 200 rpm for about one hour, then spread them on solid LB medium and culture them in a constant temperature incubator at 37 °C for 16 hours.

[0183] S2. Preparation of recombinant plasmid: ① Select well-growing single colonies and add them to liquid synthetic medium, culture them in a constant temperature shaking incubator at 37 °C and 200 rpm for 4 hours, take a small amount of bacterial liquid, extract the plasmid with a plasmid extraction kit, and then perform SalI+XbaI digestion identification; ② Expand the culture of the bacterial liquid with correct digestion identification results in a constant temperature shaking incubator at 37 °C and 200 rpm for 14 hours, and centrifuge at 4000 rpm for about 10 min to harvest the bacterial sludge; ③ Lyse the collected bacterial sludge, add Lysis Buffer I, use a shaker to fully suspend the bacteria, and the volume ratio of the bacterial sludge to Lysis Buffer I is 4:250; after the bacteria are fully suspended, add Lysis Buffer II, gently invert and mix to lyse the bacteria; add Lysis Buffer III, immediately gently invert and mix to fully neutralize it, and let it stand at room temperature for 5 min. The volume ratio of Lysis Buffer I, Lysis Buffer II and Lysis Buffer III is 5:5:7; centrifuge the lysed mixture at 12000 rpm for 5 min, take the supernatant and add it to the DNA adsorption column, centrifuge at 12000 rpm for 1 min to remove the waste liquid; add the washing solution to the adsorption column, centrifuge at 12000 rpm for 1 min to remove the waste liquid; add the elution buffer, let it stand at 37 °C for 5 min and then centrifuge at 12000 rpm for 1 min, and collect the eluate to obtain the DNA solution of the circular plasmid.

[0184] S3. Preparation of mRNA stock solution: I. Plasmid linearization: ① Use the BspQ1 digestion system and incubate at 50 °C for 1 hour to digest the circular plasmid into a linearized plasmid. Take 20 μg of the digested product for verification by agarose gel electrophoresis; ② Add 1.0 times the digested product to the reaction system, then add 0.5 times the volume of magnetic beads, and mix well. Incubate at room temperature for 10 min and then place on a magnetic stand. After the solution becomes clear, remove the supernatant; ③ Wash the magnetic beads with freshly prepared 80% ethanol solution. After removing the supernatant, dry the magnetic beads. Add nuclease-free H2O equal to 0.1 times the reaction system volume without enzyme and pipette to mix well. Then aspirate the supernatant to obtain the purified linear plasmid DNA solution, and store it at -20 °C.

[0185] The digested product is XbaI enzyme, and the manufacturer and brand from which it is purchased is Yeasen Biotech Co., Ltd. (Shanghai).

[0186] II. In Vitro Transcription Reaction IVT (In Vitro Transcription) ① Mix the linear plasmid DNA template, NTPs, CleanCap AU, and Tris-Hcl buffer as substrates, add T7 RNA polymerase, mix well, and place in a 37 °C PCR instrument for in vitro transcription reaction. After 3 hours of reaction, add DNase I to terminate the reaction; the mass ratio of the DNA template to the volume of DNase I is 1 g : 3 μL.

[0187] ② Add nuclease-free water and LiCl solution to the reaction solution. The volume ratio of the reaction solution, water, and LiCl is 1:1.5:1.5. Invert and mix well, then incubate at -20 °C for 15 min. Centrifuge at 4 °C, 12,000 g for 10 min to remove the waste liquid. Add 70% ethanol to wash the impurities. After removing the supernatant, add nuclease-free water to dissolve the precipitate to obtain the mRNA stock solution, and store it at -80 °C.

[0188] The concentration of the DNA template is 0.05 - 0.1 μg / μL.

[0189] The amount of NTPs used is 0.1 times the total volume.

[0190] The amount of CleanCap AU used is 0.1 times the total volume.

[0191] The amount of Tris-Hcl buffer used is 0.1 times the total volume.

[0192] The amount of T7 RNA polymerase used is 0.5 times the total volume.

[0193] The DNA template, NTPs, CleanCap AU, Tris-Hcl buffer, T7 RNA polymerase, and DNase I are all commercially available products.

[0194] The DNA template can be purchased from Nanjing Novoprotein Science & Technology Co., Ltd.

[0195] The NTPs can be purchased from Nanjing Novoprotein Science & Technology Co., Ltd.

[0196] The CleanCap AU can be purchased from Jiangsu Shenji Biotechnology Co., Ltd.

[0197] The Tris-Hcl buffer can be purchased from Thermo Fisher Scientific.

[0198] The T7 RNA polymerase can be purchased from Thermo Fisher Scientific.

[0199] The DNase I can be purchased from Thermo Fisher Scientific.

[0200] Encapsulation of S4.mRNA stock solution ① Thaw the mRNA stock solution in a cold water bath and dilute it with citrate buffer to a concentration of about 0.5 mg / ml to obtain the mRNA working solution; the pH value of the citrate buffer is 5.0.

[0201] ② Wrap the self-replicating mRNA in LNP to obtain the organic phase (lipid phase), and the molar concentration ratio of cations to DSPC, CHO-HP, and DMP-PEG2000 in the lipid phase is 30:5:30:2, and the total molar concentration of the lipid phase is 15 mmol / L; ③ The volume ratio of the organic phase to the mRNA working solution is 1:2, and nanoparticles LNP are prepared by a microfluidic control device; ④ Add PBS buffer with a volume 5 times that of the harvested nanoparticles for dilution, centrifuge and concentrate at 4°C and 1500 g using a 100KD ultrafiltration centrifuge tube. When the volume is concentrated to about the volume of the initially obtained LNP, add PBS buffer with a volume 5 times that of the concentrated volume for dilution, continue to concentrate to 1-0.5 times the volume before dilution, and filter through a 0.2 μm membrane to obtain the LNP finished product and the MenB preventive vaccine.

[0202] The cation type in the lipid phase can be any one of DLin-MC3-DMA, JK-0042, JK-0043, and JK-0045; the cation type used in this experiment is JK-0042.

[0203] The preventive MenB vaccine is stored at -20°C.

[0204] The pH value of the PBS buffer is 7.5.

[0205] The manufacturer from which DLin-MC3-DMA is purchased is Xiamen Syno Biological Co., Ltd.

[0206] JK-0042 is derived from Ningbo Junjian Biotechnology Co., Ltd.

[0207] JK-0043 is derived from Ningbo Junjian Biotechnology Co., Ltd.

[0208] JK-0045 is derived from Ningbo Junjian Biotechnology Co., Ltd.

[0209] The fourth group of experiments The fourth group of experiments includes Example 13, Example 14, Example 15, and Example 16.

[0210] In Example 13, the four antigen proteins, fHbp, NHBA, NadA, and PorA, of SEQ ID NO: 15 in the self-replicating mRNA sequence are designed separately, and the stock solution is mixed before encapsulation and then the preparation is carried out, which is defined as SEQ ID NO: 15-split-stock solution mixture.

[0211] In Example 14, the four antigen proteins, fHbp, NHBA, NadA, and PorA, of SEQ ID NO: 15 in the self-replicating mRNA sequence are designed separately, and they are mixed after encapsulation, and then the preparation is purified and sub-packaged, which is defined as SEQ ID NO: 15-split-preparation mixture.

[0212] In Example 15, the four antigen proteins, fHbp, NHBA, NadA, and PorA, of SEQ ID NO: 17 in the self-replicating mRNA sequence are designed separately, and the stock solution is mixed before encapsulation and then the preparation is carried out, which is defined as SEQ ID NO: 17-split-stock solution mixture.

[0213] In Example 16, the four antigen proteins, fHbp, NHBA, NadA, and PorA, of SEQ ID NO: 17 in the self-replicating mRNA sequence are designed separately, and they are mixed after encapsulation, and then the preparation is purified and sub-packaged, which is defined as SEQ ID NO: 17-split-preparation mixture.

[0214] The encapsulation method used in Example 13, Example 14, Example 15, and Example 16 includes the following steps: S1. Vector construction: The self-replicating mRNA sequence was cloned into the self-replicating vector by double digestion with SalI / XbaI to construct a self-replicating mRNA plasmid. Then, the DNA sequence of the self-replicating mRNA plasmid was introduced into Escherichia coli cells by heat shock method to establish a research and development seed bank. S2. Plasmid preparation: ① Take the engineering bacteria seeds from the research and development seed bank and add them to a liquid synthetic medium in an oscillating incubator, and measure the OD600 of the seeds to reach 0.6 - 2.5. The amplified seeds were cultured in a fermenter for 40 hours, the fermentation temperature was controlled at 30 °C, the rotation speed was 500 rpm, the dissolved oxygen was related to the stirring speed, the pH was controlled at 6.3 - 7.0, and the feeding was related to the pH. After fermentation, the supernatant was removed by centrifugation at 4000 rpm for about 5 minutes to harvest the bacterial sludge. ② Mix the bacterial sludge with resuspension solution S1 at a mass ratio of 1:10. Add solution S2 at a ratio twice that of solution S1, let it stand for 5 minutes, then add 3 times solution S3 and ammonium bicarbonate, and let it stand for 60 minutes. Remove the floating impurities and filter using a filter. Concentrate the solution using an ultrafiltration system. The pH value of solution S1 is 7.5 - 8.5 and it is composed of 50 mM glucose, 30 mM Tri-HCl and 15 mM EDTA. Solution S2 is composed of freshly prepared 0.2 M NaOH and 1% SDS. Solution S3 is a mixed solution formed by 3 M KAc and 2 M HAc.

[0215] ③ Perform chromatography using a molecular sieve chromatography column. The linear flow rate of chromatography is 3 cm / h. At about 1 / 3 CV after sample loading, the target product will pass through and the UV curve will rise, and collect this passing-through peak. The buffer solution used for chromatography with the molecular sieve chromatography column is composed of 2.0 M (NH4)2SO4, 0.5 M NaCl, 0.1 M Tris-HCl and 0.01 M EDTA-2Na.

[0216] ④ Perform chromatography using an affinity chromatography column, equilibrate with buffer solution A, and the linear flow rate of chromatography is 10 cm / h. After sample loading, use 15% buffer solution B to wash the impurities, and wash about 5 CV.

[0217] The buffer solution A is composed of 2.3 M (NH4)2SO4, 0.1 M Tris-HCl, 0.01 M EDTA-2Na; The buffer solution B is composed of 2.3 M (NH4)2SO4, 0.2 M NaCl, 0.1 M Tris-HCl, 0.01 M EDTA-2Na; and elute with 30% buffer solution B, and collect the elution peak.

[0218] ⑤ Use an ultrafiltration system to change the solution, change the salt solution to 1×TE buffer solution, and select a 300 kD3 hollow fiber column.

[0219] ⑥ Mix supercoiled plasmid DNA, linearizing enzyme, and water, incubate at 50 °C for 2 hours, and purify using anion chromatography column chromatography and ultrafiltration to obtain a linearized plasmid DNA template, which is stored at -80 °C; the linear flow rate of the chromatography is 10 - 15 cm / h, the chromatography solution A is composed of EDTA-2Na and Tris-HCl, the mass ratio of EDTA-2Na to Tris-HCl is 1.681:7.88, and the pH of the chromatography solution A is 7.5; The chromatography solution B is composed of EDTA-2Na and Tris-HCl, the mass ratio of EDTA-2Na to Tris-HCl is 1.681:7.88, and the pH of the chromatography solution B is 7.5.

[0220] Preparation of the mRNA stock solution: ① Take the linearized plasmid DNA template, NTPs, and Tris-Hcl buffer as substrates, mix them, and then add T7 RNA polymerase, and react at 33 °C for 3 hours in a synthesis workstation; ② Purify by affinity chromatography column chromatography, and select OligodT as the chromatography packing material. Dilute the reaction solution 20 times with the equilibration solution before purification, the linear purification flow rate is 10 cm / h, and wash away impurities with 5 CV. The equilibration solution is composed of NaCl, EDTA, and TrisHCl, and the mass ratio of NaCl, EDTA, and TrisHCl is 9.35:0.067:0.32; after washing away impurities, elute with pure water and collect the elution peak; ③ Purify using a TFF system, and the replacement solution is pure water. The transmembrane pressure is 10 psi, the shear rate is 3000 / sec, wash and filter 10 times and then concentrate to the target concentration. The obtained sample is filtered through a 0.2 μm filter to obtain the mRNA stock solution, which is stored at -80 °C.

[0221] The concentration of the DNA template is 0.05 - 0.1 μg / μL.

[0222] The dosage of NTPs is 0.1 times the total volume.

[0223] The dosage of the Tris-Hcl buffer is 0.1 times the total volume.

[0224] The dosage of T7 RNA polymerase is 0.5 times the total volume.

[0225] The DNA template, NTPs, Tris-Hcl buffer, and T7 RNA polymerase are all commercially available products.

[0226] The brand of the purchased manufacturer of the DNA template can be Nanjing Novoprotein Biotechnology Co., Ltd.

[0227] The purchasing manufacturer brand of the NTPs can be Nanjing Novoprotein Scientific, Inc.

[0228] The purchasing manufacturer brand of the Tris-Hcl buffer can be Thermo Fisher Scientific.

[0229] The purchasing manufacturer brand of the T7 RNA polymerase can be Thermo Fisher Scientific.

[0230] Encapsulation of S4.mRNA stock solution: ① Thaw the mRNA stock solution in a cold water bath and dilute the mRNA stock solution with citrate buffer to a concentration of about 0.5 mg / ml to obtain the mRNA working solution; the pH value of the citrate buffer is 5.0.

[0231] ② Wrap the self-replicating mRNA in LNP to obtain an organic phase (lipid phase), and the molar concentration ratio of cations to DSPC, CHO-HP, and DMP-PEG2000 in the lipid phase is 30:5:30:2, and the total molar concentration of the lipid phase is 15 mmol / L.

[0232] ③ The volume ratio of the organic phase to the mRNA working solution is 1:3, and nanoparticles LNP are prepared by a microfluidic control preparation instrument; ④ Add 1×PBS with a volume 10 times that of the harvested nanoparticles to solidify the LNP particles, and then use a TFF ultrafiltration system for purification. The replacement solution is 1×PBS; the transmembrane pressure is 10 psi, the shear rate is 2000 / sec, dilute and concentrate 6 times, wash and filter 6 cycles, and then concentrate 3 times. The obtained sample is filtered through a 0.2 μm filter to obtain the LNP-encapsulated drug composition of mRNA, and it is stored at -20°C.

[0233] The cation model in the lipid phase can be any one of DLin-MC3-DMA, JK-0042, JK-0043, and JK-0045; the cation model in the lipid phase used in this experiment is JK-0042.

[0234] Performance test: 1. Electrophoresis test: Perform gel electrophoresis tests on the optimized self-replicating mRNA sequences of Examples 1, 2, 3, 4, 5, and 6 in the second group of experiments and the recombinant plasmids obtained according to the preparation method provided in the third group of experiments to obtain plasmid identification maps.

[0235] The electrophoresis test results for the self-replicating mRNA sequence SEQIDNO:15 and its recombinant plasmid are shown in Figure 1 .

[0236] The electrophoresis test results obtained for the self-replicating mRNA sequence SEQ ID NO: 17 and its recombinant plasmid are shown in Figure 2 .

[0237] The electrophoresis test results obtained for the self-replicating mRNA sequence SEQ ID NO: 19 and its recombinant plasmid are shown in Figure 3 .

[0238] The electrophoresis test results obtained for the self-replicating mRNA sequence SEQ ID NO: 21 and its recombinant plasmid are shown in Figure 4 .

[0239] The electrophoresis test results obtained for the self-replicating mRNA sequence SEQ ID NO: 23 and its recombinant plasmid are shown in Figure 5 .

[0240] The electrophoresis test results obtained for the self-replicating mRNA sequence SEQ ID NO: 25 and its recombinant plasmid are shown in Figure 6 .

[0241] 2. Cell experiments: (1) Collection of cell protein samples: ① Culture HEK293 cells on a cell culture plate until the cell confluence in each well reaches 80%-90%; ② Take 2 sterile EP tubes respectively. Add 2-5 μg of mRNA sample to one tube and dilute it to 150 μL with Opti-MEM (serum-free medium). Add 3-5 μL of transfection reagent Lipofectamine™ MessengerMAX™ Reagent to the other tube and also make up to 150 μL with Opti-MEM. Mix the two tubes of liquid by pipetting, let it stand for 5 min, and then add the standing solution dropwise into the well plate and culture it in a carbon dioxide incubator for 24 hours; ③ Take out the cell well plate, observe the cell state under the microscope, then place it on ice, collect the culture medium in the well, and add 1% PMSF according to the volume, mix well and place it on ice; ④ After sucking out the culture medium, wash the cell well plate once with ice-bathed 1×PBS, add 200 μL of RIPA lysis buffer to each well, add 1% PMSF to the lysis buffer, place it on ice for lysis for 5-10 min, collect the lysis buffer and vortex; ⑤ Centrifuge the collected culture medium and lysis buffer at 4°C, 17000g for 15-30 min, collect the supernatant to obtain cell protein samples, and store them at -20°C.

[0242] The SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 sequences provided in Example 7, Example 8, Example 9, Example 10, Example 11 and Example 12, as well as the positive and blank control samples, were detected for protein bands by immunoblotting. The test results are shown in Figure 7 .

[0243] The test method includes: ① According to the concentration of the cell protein sample, add 4×LDS sample buffer and 1M DTT (10×), add deionized water to make up to a 60 μl system, incubate at 90 °C for 5 - 10 min, centrifuge and mix well before loading; ② Electrophoresis: The stacking gel was electrophoresed at a constant voltage of 80 V for 20 - 30 min. The concentration of the polyacrylamide separating gel was 6%. The electrophoresis condition of the separating gel was 150 V constant voltage electrophoresis for 35 min. Stop electrophoresis after the dye reaches the bottom of the separating gel; ③ Electrotransfer: Place the PVDF membrane in the activation solution for activation; take out the gel, cut off the excess part of the gel, and make a sandwich structure in 1× transfer buffer: sponge - filter paper - gel - PVDF membrane - filter paper - sponge, ensure that there are no bubbles in the sandwich structure, add an appropriate amount of 1× transfer buffer into the transfer tank, and place it in an ice box, electrotransfer at a constant current of 200 mA for 100 min; ④ Blocking: Prepare 5% BSA with 1×TPBT as the blocking solution, put the transferred PVDF membrane into it, and incubate at room temperature on a shaker for 60 min; rinse 3 times with 1×TBST buffer, 10 min each time; ⑤ Primary antibody incubation: Dilute the primary antibody according to the instructions with 1× antibody diluent, incubate at room temperature with shaking for 1 hour, then incubate overnight at 4 °C, and restore to room temperature and incubate with shaking for 1 hour the next day; wash 3 times with 1×TBST, 5 min each time; ⑥ Secondary antibody incubation: Dilute the secondary antibody according to the instructions with 1× antibody diluent, incubate at 37 °C in the dark on a shaker for 1 hour, and then incubate at room temperature on a shaker for 20 min; wash 3 times with 1×TBST, 5 min each time; ⑦ Development: Mix the developing solutions A and B evenly in a ratio of 1:1, drop it onto the membrane, and place it in a chemiluminescent imaging system for imaging.

[0244] Figure 7The Marker in the middle is the protein molecular weight standard, which contains specific molecular weight markers such as 245 kD and 180 kD, etc., and is used to indicate the molecular weight of proteins in the sample; Lane 1 is marked as blank, which is a blank control sample. There should be no specific bands of the target protein theoretically, and it is used to detect the background signal of the experimental system and exclude interference factors such as non-specific binding; Lane 2 is marked as positive, which is a positive control sample, containing a sample with known expression of the target protein, and characteristic bands of the target protein will appear, and it is used to verify the effectiveness of the experimental system and ensure that the experimental procedure can correctly detect the target protein; Lane 3 - Lane 8 are the detection bands of samples related to the sequences of SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 respectively; By comparing with the Marker, the molecular weight of the target protein in the sample can be judged; By comparing with the positive control, the expression of the target protein in these samples can be evaluated, including whether the target protein is successfully expressed and the relative level of expression. Information such as the position and gray scale of the bands can provide a basis for analyzing the characteristics of the target protein in each sample.

[0245] 4. Animal experiments and detection of serum-binding antibodies: 4.1 Animal experiments: ① Grouping principle: Balb / c mice were grouped according to the random grouping method, with 5 mice in each group; Each group was injected with the preventive MenB vaccine of the mRNA sequence, and the day of grouping was Day 0.

[0246] ② Animal administration: Different control groups were set up, and the prepared self-replicating mRNA-LNP samples were administered at a dose of 15 μg / mouse. The empty LNP group was used as the control group, and the first and second immunizations were administered on Day 0 and Day 21 respectively.

[0247] ③ Recording of test indicators: Observe the animals every day and record the clinical symptoms of the animals. The clinical symptoms of the animals include but are not limited to 0 normal, 1 reduced activity, 2 restlessness, 3 tremors, 4 spinning or backward movement, 5 piloerection, 6 abnormal breathing, 7 arched back, 8 significant decrease in body temperature, 9 hair loss, 10 abnormal eyes, 11 abdominal distension, 12 others.

[0248] ④ Sampling: Blood was collected from the retro-orbital area on Day 0, Day 13, Day 20, Day 29, Day 34, Day 50, D72, and Day 90 respectively.

[0249] ⑤ Termination of the experiment: After all the administration observations were completed, continue to observe for 1 - 2 months, and then the experiment was terminated.

[0250] 4.2 Detection of serum-binding antibodies: The self-replicating mRNA sequences provided in Example 1 is SEQ ID NO: 15, the self-replicating mRNA sequence provided in Example 2 is SEQ ID NO: 17, the self-replicating mRNA sequence provided in Example 3 is SEQ ID NO: 19, the self-replicating mRNA sequence provided in Example 4 is SEQ ID NO: 21, the self-replicating mRNA sequence provided in Example 5 is SEQ ID NO: 23, and the self-replicating mRNA sequence provided in Example 6 is SEQ ID NO: 25. They are respectively combined with fHbp, NHBA, NadA or PorA antigen proteins for serum binding antibody detection. The test results are shown in Figures 8 - 11 . Figure 8 It is a graph of the detection results of fHbp-binding antibodies; Figure 9 It is a graph of the detection results of NHBA-binding antibodies; Figure 10 It is a graph of the detection results of NadA-binding antibodies; Figure 11 It is a graph of the detection results of PorA-binding antibodies.

[0251] 1 μg, 5 μg, and 15 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1 are taken respectively; 1 μg, 5 μg, and 15 μg of the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2 are taken respectively; 1 μg, 5 μg, and 15 μg of the SEQ ID NO: 15-split-stock solution mixture provided in Example 13 are taken respectively; 1 μg, 5 μg, and 15 μg of the split-formulation mixture provided in Example 14 are taken respectively; 1 μg, 5 μg, and 15 μg of the SEQ ID NO: 17-split-stock solution mixture provided in Example 15 are taken respectively; 1 μg, 5 μg, and 15 μg of the split-formulation mixture provided in Example 16 are respectively combined with fHbp, NHBA, NadA or PorA antigen proteins for serum binding antibody detection. The test results are shown in Figures 16 - 19 .

[0252] Detection method: ① Dilute the MenB (fHbp) or MenB (NHBA) or MenB (NadA) or MenB (PorA) antigen with ELISA coating buffer to 1 μg / mL. Add the diluted antigen into the wells of the enzyme-linked immunosorbent assay (ELISA) plate, 100 μL / well, and coat overnight at 4 °C (14 - 18 h); ② The next day, rinse 3 times with PBST, 5 min each time. After patting dry the moisture, add 200 μL of 1% BSA to each well for blocking. Incubate at 37 °C for 1 hour, then rinse 3 times with PBST, 5 min each time, and pat dry the moisture; ③ Add the corresponding diluted mouse serum, 100 μL / well. Make 1 duplicate well for each sample, and set negative / positive controls simultaneously. Incubate at 37 °C for 1 hour, then rinse 3 times with PBST, 5 min each time, and pat dry the moisture; ④ Add anti-mouse IgG heavy chain antibody (HRP-labeled), 100 μL / well. After incubating at 37 °C for 1 hour, rinse 3 times with PBST, 5 min each time, and pat dry the moisture. ⑤ Add TMB chromogenic solution for color development, 100 μL / well. After incubating at room temperature in the dark for 10 - 15 min, add the stop solution to terminate the reaction, 50 μL / well. Measure the absorbance value using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm, and calculate the titer of each binding antibody in the sample through the OD value.

[0253] Figure 16 In it, a is the result of serum binding antibody detection when binding to PorA antigen protein with the dosage of 1 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17-split-stock solution mixture provided in Example 15, and the split-formulation mixture provided in Example 16.

[0254] Figure 16 In it, b is the result of serum binding antibody detection when binding to PorA antigen protein with the dosage of 5 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17-split-stock solution mixture provided in Example 15, and the split-formulation mixture provided in Example 16.

[0255] Figure 16 In it, c is the result of serum binding antibody detection when binding to PorA antigen protein with the dosage of 15 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17-split-stock solution mixture provided in Example 15, and the split-formulation mixture provided in Example 16.

[0256] Through Figure 16 It can be seen that the binding antibody titers of each sample group are generally low at 14 days and 21 days after administration; over time, at 35 days, 49 days, and 70 days after administration, the antibody titers of some sample groups increase significantly, reflecting the differences in the ability and time course of vaccines with different sequences, doses, and formulation forms to induce the production of MenB (porA) binding antibodies, which can be used to evaluate the immune effect of vaccines and optimize vaccine design.

[0257] Figure 17Among them, a is the result of serum binding antibody detection when binding to NadA antigen protein with the dosage of 1 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17-split-stock solution mixture provided in Example 15, and the split-formulation mixture provided in Example 16.

[0258] Figure 17 Among them, b is the result of serum binding antibody detection when binding to NadA antigen protein with the dosage of 5 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17-split-stock solution mixture provided in Example 15, and the split-formulation mixture provided in Example 16.

[0259] Figure 17 Among them, c is the result of serum binding antibody detection when binding to NadA antigen protein with the dosage of 15 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17-split-stock solution mixture provided in Example 15, and the split-formulation mixture provided in Example 16.

[0260] Through Figure 17 It can be seen that at 14 days and 21 days after administration, the binding antibody titers of most sample groups were at a relatively low level, indicating that the immune response of the body to the vaccine had not been fully initiated at this time. At 35 days after administration, the antibody titers of some sample groups began to increase significantly, showing that the immune response of the body gradually enhanced. At 49 days and 70 days after administration, the antibody titers of multiple sample groups reached a relatively high level, indicating that there were differences in the ability and time course of vaccines with different sequences, dosages, and formulation forms to induce the production of MenB (NadA) binding antibodies.

[0261] Figure 18 Among them, a is the result of serum binding antibody detection when binding to NHBA antigen protein with the dosage of 1 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17-split-stock solution mixture provided in Example 15, and the split-formulation mixture provided in Example 16.

[0262] Figure 18In b, the results of serum binding antibody detection by binding to NHBA antigen protein are shown when the dosages of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17-split-stock solution mixture provided in Example 15, and the split-formulation mixture provided in Example 16 are 5 μg.

[0263] Figure 18 In c, the results of serum binding antibody detection by binding to NHBA antigen protein are shown when the dosages of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17-split-stock solution mixture provided in Example 15, and the split-formulation mixture provided in Example 16 are 15 μg.

[0264] Through Figure 18 It can be seen that on the 14th and 21st days after administration, the binding antibody titers of each sample group are generally low, indicating that the body's immune response is weak at this time; on the 35th day after administration, the antibody titers of some sample groups begin to increase significantly; on the 49th and 70th days after administration, the antibody titers of multiple sample groups reach relatively high levels, indicating that there are differences in the ability and time course of vaccines with different sequences, dosages, and formulation forms to induce the production of MenB (NHBA) binding antibodies.

[0265] Figure 19 In a, the results of serum binding antibody detection by binding to fHbp antigen protein are shown when the dosages of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17-split-stock solution mixture provided in Example 15, and the split-formulation mixture provided in Example 16 are 1 μg.

[0266] Figure 19 In b, the results of serum binding antibody detection by binding to fHbp antigen protein are shown when the dosages of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17-split-stock solution mixture provided in Example 15, and the split-formulation mixture provided in Example 16 are 5 μg.

[0267] Figure 19Among them, c is the result of serum binding antibody detection when binding to the fHbp antigen protein with the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17-split-stock solution mixture provided in Example 15, and the split-formulation mixture provided in Example 16 at a dosage of 15 μg.

[0268] Through Figure 19 It can be seen that 14 days and 21 days after administration, the binding antibody titers of each sample group were generally low, indicating that the immune response of the body was weak at this time; 35 days after administration, the antibody titers of some sample groups began to increase significantly; 49 days and 70 days after administration, the antibody titers of multiple sample groups reached relatively high levels, indicating that there were differences in the ability and time course of vaccines with different sequences, doses, and formulation forms to induce the production of MenB (fHbp) binding antibodies.

[0269] 4.3. Immunocyte typing detection (FACs): Take the spleens of mice injected with the preventive MenB vaccines provided in Example 7, Example 8, Example 9, Example 10, Example 11, and Example 12 to examine T cell activation; after the spleen cells are stimulated with polypeptides, they are stained with IFN-γ / CD69 dyes and then analyzed for T cell activation by the FACs method. The test results at 34 days and 70 days after injection are shown in Figures 12 - 13 .

[0270] Figure 12 It is a FACs detection result diagram at D34 days after injecting the preventive MenB vaccine.

[0271] Figure 13 It is a FACs detection result diagram at D70 days after injecting the preventive MenB vaccine.

[0272] Respectively inject 1 μg, 5 μg, and 15 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1 into mice; respectively inject 1 μg, 5 μg, and 15 μg of the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2 into mice; respectively inject 1 μg, 5 μg, and 15 μg of the SEQ ID NO: 15-split-stock solution mixture provided in Example 13 into mice; respectively inject 1 μg, 5 μg, and 15 μg of the split-formulation mixture provided in Example 14 into mice; respectively inject 1 μg, 5 μg, and 15 μg of the SEQ ID NO: 17-split-stock solution mixture provided in Example 15 into mice; respectively inject 1 μg, 5 μg, and 15 μg of the split-formulation mixture provided in Example 16 into mice, and record the immunocyte typing detection results after injection. The test results are shown in Figures 20 - 23 .

[0273] Figure 20 are the detection results of CD4+IFNg+ T cell typing; Figure 21 are the detection results of CD4+CD69+ T cell typing; Figure 22 CD8+IFNg+ T cell typing detection results; Figure 23 are the detection results of CD8+CD69+ T cell typing.

[0274] Through Figures 20 - 23 it can be seen that after administration of vaccines from different samples, they can all induce the activation of CD4⁺ and CD8⁺ T cell subsets and changes in function-related indicators to a certain extent; the frequencies of CD4⁺IFNγ⁺T, CD4⁺CD69⁺T, CD8⁺IFNγ⁺T, and CD8⁺CD69⁺T cells in different sample groups all increase, indicating that the vaccine can activate the body's T cell immune response, promote T cells to secrete cytokines or enter the early activation state, and participate in immune defense. There are differences in the ability and time course of inducing T cell immune responses among vaccine samples with different doses of SEQ ID NO.15 sequence and SEQ ID NO.17 sequence, as well as different doses of split-stock mixture and split-formulation mixture; as the number of days after administration increases, from 35 days to 70 days, the T cell immune response indicators induced by most sample groups show an increasing trend, reflecting that the body's cellular immune response to the vaccine is a dynamic process and gradually enhances within a certain period after administration.

[0275] 4.4. Detection of IFN-γ cytokine secretion level (ELISOPT): Take the spleen cells of the mice vaccinated with the preventive MenB vaccine provided in Injection Example 7, Injection Example 8, Injection Example 9, Injection Example 10, Injection Example 11, and Injection Example 12 and use the ELISPOT method to detect the secretion of IFN-γ. Prepare a mouse cell suspension from the spleen cells, add a polypeptide for stimulation to make the spleen cells secrete IFN-γ, add an avidin-enzyme complex, bind it to biotin, add a chromogenic substrate to form visible spots, and record the ELISOPT test results at 34 days and 70 days after injection as shown in Figures 14 - 15 .

[0276] Figure 14 is the ELISPOT detection result chart at D34 days after injection of the preventive MenB vaccine.

[0277] Figure 15 is the ELISPOT detection result chart at D70 days after injection of the preventive MenB vaccine.

[0278] 1 μg, 5 μg, and 15 μg of the self-replicating mRNA sequence SEQ ID NO:15 provided in Example 1 were respectively injected into mice; 1 μg, 5 μg, and 15 μg of the self-replicating mRNA sequence SEQ ID NO:17 provided in Example 2 were respectively injected into mice; 1 μg, 5 μg, and 15 μg of the SEQ ID NO:15-split-stock solution mixture provided in Example 13 were respectively injected into mice; 1 μg, 5 μg, and 15 μg of the SEQ ID NO:15 split-formulation mixture provided in Example 14 were respectively injected into mice, and the ELISOPT test results after injection were recorded as shown in Figures 24 - 25 .

[0279] Figure 24 The ELISPOT test results on the 35th day after injection with D; Figure 25 The ELISPOT test results on the 70th day after injection with D.

[0280] Through Figure 7 The results of detecting protein bands by Western Blotting provided showed that the 6 sequences of SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, and SEQ ID NO:25 provided in Example 7, Example 8, Example 9, Example 10, Example 11, and Example 12 all showed target bands at the corresponding positions, indicating that the above 6 sequences could all normally express the target protein in cells. Figures 8 - 11 The results of binding antibodies showed that the antibody titers of the SEQ ID NO:15 group and the SEQ ID NO:17 group were the highest. Figures 12 - 13 showed that there were no significant differences in the immune cell typing of each group in the FACs test results after injection with the MenB vaccine prophylaxis in Figures 14 - 15 showed that the ELISPOT results after injection with the MenB vaccine prophylaxis were slightly higher in the SEQ ID NO:17 group and the SEQ ID NO:19 group. From the test results presented by the intracellular protein expression, in vivo binding antibody data, FACs test data, and ELISPOT results, it can be seen that the self-replicating mRNA sequences designed in this application can induce an immune response against Neisseria meningitidis in vivo, and the experimental effect of the SEQ ID NO:17 group is the best.

[0281] Figure 24In a, the ELISPOT detection results on day D35 after injecting into mice at a dosage of 1 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15 split-formulation mixture provided in Example 14.

[0282] Figure 24 In b, the ELISPOT detection results on day D35 after injecting into mice at a dosage of 5 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15 split-formulation mixture provided in Example 14.

[0283] Figure 24 In c, the ELISPOT detection results on day D35 after injecting into mice at a dosage of 5 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15 split-formulation mixture provided in Example 14.

[0284] Figure 25 In a, the ELISPOT detection results on day D70 after injecting into mice at a dosage of 1 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15 split-formulation mixture provided in Example 14.

[0285] Figure 25 In b, the ELISPOT detection results on day D70 after injecting into mice at a dosage of 5 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15 split-formulation mixture provided in Example 14.

[0286] Figure 25Among them, c is the ELISPOT test result on the 70th day after injecting 5 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15-split-formulation mixture provided in Example 14 into mice.

[0287] Through Figure 24 and Figure 25 It can be seen that on the 35th day and the 70th day, a certain number of cells secreting cytokines (reflected by the SFU value) were detected in different sample groups under the stimulation of multiple antigens (fHbp, NHBA, NadA, PorA), indicating that vaccine samples with different sequences, doses, and formulation forms can activate immune cells in the body to a certain extent, prompting them to secrete cytokines and initiate an immune response.

[0288] 4.5 Serum bactericidal titer detection: Cultivate the revived strain in an environment of 37°C and 5% CO2 for 16 - 24 h. Take about 20 - 40 colonies for subculture, incubate in an environment of 37°C and 5% CO2 for 4 h to prepare a bacterial suspension, and adjust it to an appropriate concentration. Inactivate the serum to be tested, and perform serial dilutions with bactericidal buffer in a 96-well U-bottom microtiter plate. Add the SEQ ID NO: 15-split-formulation mixture and the SEQ ID NO: 17-split-formulation mixture as complement and bacterial suspension to the diluted serum in sequence, and add the corresponding complement SEQ ID NO: 15-split-formulation mixture or SEQ ID NO: 17-split-formulation mixture and bacterial suspension to the complement control and serum control wells. Seal the microtiter plate and incubate it at 37°C for 60 minutes. Take 10 μL from each well and inoculate it onto a Columbia blood agar plate using the tilting method. Incubate the plate in an environment of 37°C and 5% CO2 overnight and count the colonies. The bactericidal titer of the sample to be tested is the highest dilution factor with the number of colonies less than 50% of the live complement well as a reference; the test result of the SEQ ID NO: 17-split-formulation mixture is shown in Figure 26 ; the test result of the SEQ ID NO: 15-split-formulation mixture is shown in Figure 27 .

[0289] From Figure 26 it can be seen that the serum bactericidal titer corresponding to the SEQ ID NO: 17-split-formulation mixture is 1:32, which means that when the serum is diluted to 1:32, it can still effectively kill a certain number of bacteria. Above this dilution, the bactericidal ability decreases and the number of colonies on the plate increases significantly.

[0290] From Figure 27It can be seen from [SEQ ID NO: 15 split - formulation mixture] that the serum bactericidal titer corresponding to it is 1:256, which means that when the serum is diluted to 1:256, it can still effectively kill a certain number of bacteria. Above this dilution, the bactericidal ability weakens and the number of colonies on the plate increases.

Claims

1. A self-replicating mRNA sequence, characterized in that, RNA comprising the coding region in the amino acid sequence, said amino acid sequence comprising a tandem sequence formed by concatenating the individual sequences shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 in any order before and after.

2. The self-replicating mRNA sequence according to claim 1, wherein The amino acid sequence is optimized for fHbp, NHBA, NadA and PorA antigen proteins and the original signal peptide sequence is deleted.

3. The self-replicating mRNA sequence according to claim 1, characterized in that, One or more signal peptide sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 are added at the 5'-end of the tandem sequence.

4. The self-replicating mRNA sequence according to claim 3, wherein The individual sequences in the tandem sequence are linked using the flexible Linker in SEQ ID NO:11 in the amino acid sequence.

5. The self-replicating mRNA sequence according to claim 4, wherein The MITD molecular sequence is added at the end of the tandem sequence, and the MITD molecular sequence comprises the amino acid sequence SEQ ID NO:12 or SEQ ID NO:

13.

6. The self-replicating mRNA sequence according to claim 1, wherein, The self-replicating mRNA sequence comprises the target protein amino acids designed by molecular concatenation for fHbp, NHBA, NadA and PorA antigen proteins.

7. The self-replicating mRNA sequence according to claim 1, characterized in that, The stop codon of the self-replicating mRNA sequence comprises the amino acid sequence SEQ ID NO:

26.

8. The self-replicating mRNA sequence according to claim 1, characterized in that, The self-replicating mRNA sequence comprises the amino acid sequence shown in SEQ ID NO:

17.

9. A MenB vaccine, characterized in that, Comprising the self-replicating mRNA sequence according to any one of claims 1-8.

10. A method for preparing a MenB vaccine as claimed in claim 9, characterized in that, Comprising the following steps: Vector construction, plasmid preparation, preparation of the mRNA stock solution, encapsulation of the mRNA stock solution, to obtain a prophylactic MenB vaccine.

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