CN120350043B

CN120350043B - A self-replicating mRNA sequence and a vaccine for preventing MenB and a preparation method thereof

Info

Publication number: CN120350043B
Application number: CN202510797659.2A
Authority: CN
Inventors: 熊长云; 李智; 王友如; 陈庚; 周芮; 邢瑞; 王学莲; 冉丽
Original assignee: Ningbo Junjian Biotechnology Co ltd
Current assignee: Ningbo Junjian Biotechnology Co ltd
Priority date: 2025-06-16
Filing date: 2025-06-16
Publication date: 2025-09-19
Anticipated expiration: 2045-06-16
Also published as: CN120350043A

Abstract

The application relates to the technical field of MenB vaccines, in particular to a self-replicating mRNA sequence, a MenB vaccine for preventing and a preparation method thereof. A self-replicating mRNA sequence comprises RNA of a coding region in an amino acid sequence, wherein the amino acid sequence comprises a tandem sequence formed by tandem connection of single sequences shown in SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 or SEQ ID NO. 10 according to any sequence. The application provides a MenB vaccine for preventing infection and reducing mortality caused by B type meningococcus aiming at strains discovered in China monitoring of coverage, and the MenB vaccine has the advantages of low injection needle number, low dosage and high expression effect, can reduce dosage and frequency, and improves the expression efficiency of the vaccine so as to achieve better immune effect.

Description

Self-replicating mRNA sequence, menB-preventing vaccine and preparation method

Technical Field

The invention relates to the technical field of MenB vaccines, in particular to a self-replicating mRNA sequence, a MenB vaccine for preventing and a preparation method thereof.

Background

Epidemic cerebrospinal meningitis is triggered by neisseria meningitidis, type B meningitis has been popular in recent years in our country and in many countries, infants and children under 5 years of age being high risk populations. The current type B meningococcal vaccine does not cover the epidemic strains in China, so that the research and development of the vaccine for effectively preventing the type B meningitis plays an important role in prevention and control.

The recombinant protein vaccine production has the problems of complex process, high cost, difficult purification, time consumption and the like, and influences the public acceptance. Therefore, providing a highly efficient and safe self-replicating mRNA molecule and preventing MenB vaccine is a major technical problem that needs to be solved at present.

Disclosure of Invention

The patent designs a self-replicating mRNA vaccine aiming at four antigens of type B meningitis, adopts lipid nano particles to wrap, and codes serial sequences 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 generate four antigens aiming at B-type meningitis, and has wide application prospect.

The inventor finds that an mRNA vaccine sequence for preventing the MenB vaccine from generating specific immune response, high immunogenicity, lasting immune response and low vaccine cost is provided by utilizing the self-replicating mRNA sequence in the experimental process.

The first aspect of the invention provides a self-replicating mRNA sequence, which comprises RNA of a coding region in an amino acid sequence, wherein the amino acid sequence comprises a serial sequence formed by serially connecting single sequences shown in SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 or SEQ ID NO. 10 in any order.

For strains found in China monitoring of coverage, the protein sequences of the application cover the ST-4821, ST-41/44, ST-32, ST-198, ST-175 clone complexes in Neisseria meningitidis serogroup B bacteria. The amino acid sequence of the Chinese strain is screened, the original signal peptide sequence is optimized and deleted for the fHbp and NHBA, nadA, porA antigen proteins, and the optimized protein amino acid sequence comprises SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 or SEQ ID NO. 10.

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.

In one embodiment, the fHbp antigen protein molecule deletes the pro 1-19 signal peptide sequence as amino acid sequence seq id No. 7.

In one embodiment, the NHBA antigen protein molecule deleter 1-17 signal peptide sequence is used as the amino acid sequence SEQ ID NO:8.

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

In one embodiment, the PorA antigen protein molecule deleter 1-18 signal peptide sequence is used as the amino acid sequence SEQ ID NO:10.

In one embodiment, the 5' end of the tandem sequence is added with 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.

In one embodiment, the amino acid sequence is a tandem sequence formed by tandem connection of SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 or SEQ ID NO.10 according to any sequence.

The inventor finds in the experimental process that based on the design of the Chinese strain sequence, the constructed series sequence can be ensured to more accurately match with the characteristics of the MenB strain popular in China, thereby laying a foundation for the effectiveness of the MenB vaccine. The inventors have found during the course of experiments that different alignment sequences may affect the spatial structure and immunogenicity of the final protein, and that through the possibility of multiple alignment combinations, more choices are provided for screening the most immunologically active amino acid sequences.

In one embodiment, the signal peptide sequence added at the 5' end of the tandem sequence comprises a novel coronavirus signal peptide, and the sequence is SEQ ID NO:1.

In one embodiment, the signal peptide sequence added at the 5' end of the tandem sequence comprises the tPA signal peptide, and the sequence is SEQ ID NO:2.

In one embodiment, the signal peptide sequence added at the 5' end of the tandem sequence comprises a fHbp signal peptide with the sequence of seq id no:3.

In one embodiment, the signal peptide sequence added at the 5' end of the tandem sequence comprises NHBA signal peptide with the sequence of SEQ ID NO. 4.

In one embodiment, the signal peptide sequence added at the 5' end of the tandem sequence comprises an NadA signal peptide with the sequence of seq id No. 5.

In one embodiment, the signal peptide sequence added at the 5' end of the tandem sequence comprises a PorA signal peptide with the sequence of SEQ ID NO:6.

The inventor further optimizes the tandem sequence, adds a signal peptide sequence at the 5' end of the tandem sequence, guides the new-born protein to enter a specific secretion path in cells, ensures that the protein can be correctly positioned and function, ensures the secretion of self-replicase and antigen, and discovers that different signal peptides can have different guiding characteristics in the experimental process, thereby being applicable to different cellular environments and protein functional requirements.

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

To further optimize the tandem sequence, a signal peptide sequence shown in SEQ ID NO:1 was added at the 5' end of the tandem sequence. The inventor finds that the SEQ ID NO:1 signal peptide is most excellent in guiding the protein encoded by the tandem sequence to enter the secretory pathway in the experimental process, and can remarkably improve the expression efficiency and correct positioning rate of the protein, thereby enhancing the immunogenicity of the vaccine.

In one embodiment, the individual sequences of the tandem sequence are linked using the amino acid sequence SEQ ID NO:11 flexible Linker.

In one embodiment, the tandem sequences are linked between individual sequences using a flexible Linker as shown in SEQ ID NO. 11.

In one embodiment, the amino acid sequence is a tandem sequence formed by tandem connection of SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 or SEQ ID NO. 10 according to any sequence, a signal peptide sequence shown as 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 flexible Linker shown as SEQ ID NO. 11 is used for connection between the single sequences in the tandem sequence.

In one embodiment, the amino acid sequence is a tandem sequence formed by tandem connection of SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 or SEQ ID NO. 10 according to any sequence, a signal peptide sequence shown as SEQ ID NO.1 is added at the 5' end of the tandem sequence, and flexible Linker shown as SEQ ID NO.11 is used for connection between the single sequences in the tandem sequence.

The inventor finds that in the experimental process, in order to ensure that protein domains coded by individual sequences in the tandem sequence can independently play a role, avoid steric hindrance and interference between each other, and limit the tandem sequence to use flexible Linker connection shown as SEQ ID NO:11 between the individual sequences, enough space freedom degree can be provided for each protein domain, so that each protein domain can be freely folded and moved, and thus the correct conformation and functional integrity of the whole tandem protein are ensured.

In one embodiment, MITD molecules of sequence are added at the tail end of the tandem sequence, and the MITD molecules of sequence comprise amino acid sequence SEQ ID NO. 12 or SEQ ID NO. 13.

In one embodiment, the MITD sequence is preferably SEQ ID NO. 12, and the MITD sequence designed by adding mutation is more preferably SEQ ID NO. 13.

The inventor finds that MITD is a special sequence which can be added to vaccine antigens in experiments and can guide the antigens to enter specific areas of dendritic cells, so that the antigens are easier to be processed by the dendritic cells and presented to T cells, thereby starting immune response.

The inventors found in experiments for the mutant design that the wild-type sequence based on antigen fHbp was SEQ ID NO:27 and the mutated sequence based on antigen fHbp was SEQ ID NO:28.

The inventors found in experiments that the wild-type sequence SEQ ID NO:29 based on antigen NHBA and the mutated sequence SEQ ID NO:30 based on antigen NHBA were obtained.

The inventors found in experiments for the mutant design that 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.

The inventors found in experiments that the wild-type sequence SEQ ID NO:33 based on the antigen PorA and the mutated sequence SEQ ID NO:34 based on the antigen PorA were obtained.

In one embodiment, the self-replicating mRNA sequence includes target protein amino acids designed in tandem for fHbp, NHBA, nadA, and PorA antigen proteins.

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

In one embodiment, the molecular tandem design is performed for four antigenic proteins, fHbp, NHBA, nadA, porA, as self-replicating mRNA sequences.

In one embodiment, the novel coronavirus SP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA target protein amino acid is obtained by molecular tandem design of the four antigen proteins of fHbp, NHBA, nadA, porA, and the target protein amino acid sequence is SEQ ID NO:14.

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

In one embodiment, the tPASP-fHbp-Linker-NHBA-Linker-NadA-Linker-porA target protein amino acid is obtained by molecular tandem design of the four antigen proteins fHbp, NHBA, nadA, porA, and the target protein amino acid sequence is SEQ ID NO:16.

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

In one embodiment, the fHbpSP-fHbp-Linker-NHBA-Linker-NadA-Linker-porA target protein amino acid is obtained by molecular tandem design of the four antigen proteins fHbp, NHBA, nadA, porA, and the target protein amino acid sequence is SEQ ID NO:18.

In one embodiment, the GC content and the secondary structure of the target protein are optimized by adjusting the codon sequence of the amino acid sequence SEQ ID NO:18, and the optimized base sequence is SEQ ID NO:19.

In one embodiment, the NHBASP-fHbp-Linker-NHBA-Linker-NadA-Linker-porA target protein amino acid is obtained by molecular tandem design of the four antigen proteins fHbp, NHBA, nadA, porA, and the target protein amino acid sequence is SEQ ID NO:20.

In one embodiment, the GC content and the secondary structure of the target protein are optimized by adjusting the codon sequence of the amino acid sequence SEQ ID NO:20 of the target protein, so that the optimized base sequence is SEQ ID NO:21.

In one embodiment, the NadASP-fHbp-Linker-NHBA-Linker-NadA-Linker-porA target protein amino acid is obtained by molecular tandem design of the four antigen proteins fHbp, NHBA, nadA, porA, and the target protein amino acid sequence is SEQ ID NO:22.

In one embodiment, the GC content and the secondary structure of the target protein are optimized by adjusting the codon sequence of the target protein amino acid sequence SEQ ID NO:22, and the optimized base sequence is SEQ ID NO:23.

In one embodiment, the PorASP-fHbp-Linker-NHBA-Linker-NadA-Linker-porA target protein amino acid is obtained by molecular tandem design of the four antigen proteins fHbp, NHBA, nadA, porA, and the target protein amino acid sequence is SEQ ID NO:24.

In one embodiment, the GC content and the secondary structure of the target protein are optimized by adjusting the codon sequence of the target protein amino acid sequence SEQ ID NO:24, and the optimized base sequence is SEQ ID NO:25.

In one embodiment, the sequence stop codon of the self-replicating mRNA comprises the amino acid sequence SEQ ID NO. 26.

The inventors have found during experiments that in order to achieve precise control of protein translation, the synthesis of the protein can be terminated by adding a stop codon, thereby producing the desired protein variant or avoiding the production of unwanted proteins. In particular, the sequence of the termination codon is designed as SEQ ID NO. 26.

The inventor finds that in the experimental process, in order to improve the gene expression level, a high-frequency synonymous codon is selected according to the codon usage preference of a host cell, the stability of the secondary structure of an mRNA sequence is closely related to the half life period of the mRNA sequence and the protein expression quantity, the stability of the secondary structure of the mRNA sequence is enhanced, the expression of a target protein is facilitated, the stability and the translation efficiency of the mRNA sequence can be improved due to high content of guanine and cytosine (G and C), the GC content in the codon sequence of a target gene can be adjusted, and the expression level of the target gene in the host cell can be improved. By adjusting the GC content of the target gene codon sequence, the GC content 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 is 58.85%, 58.34%, 58.58%, 58.10%, 58.88% and 58.77% respectively.

In one embodiment, the self-replicating mRNA sequence includes one or more of the 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.

In one embodiment, the self-replicating mRNA sequence is the sequence shown as SEQ ID NO. 15.

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

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

In one embodiment, the self-replicating mRNA sequence is the sequence shown as SEQ ID NO. 21.

In one embodiment, the self-replicating mRNA sequence is the sequence shown as SEQ ID NO. 23.

In one embodiment, the self-replicating mRNA sequence is the sequence shown as SEQ ID NO. 25.

In one embodiment, the self-replicating mRNA sequence includes the amino acid sequence shown in SEQ ID NO. 17.

In a second aspect the invention provides a prophylactic MenB vaccine comprising a self-replicating mRNA sequence.

In a third aspect, the invention provides a method for preparing a prophylactic MenB vaccine, comprising the steps of:

vector construction, plasmid preparation, preparation of mRNA stock solution and encapsulation of the mRNA stock solution, thus obtaining the MenB vaccine.

In one embodiment, the method of preparing the prophylactic MenB vaccine comprises the steps of:

s1, constructing a vector, namely cloning a self-replication mRNA sequence to the self-replication vector through SalI/XbaI double enzyme digestion to construct a self-replication mRNA plasmid, and then introducing the DNA sequence of the self-replication mRNA plasmid into an escherichia coli cell by adopting a thermal shock method to establish a seed library;

S2, plasmid preparation:

① Fermenting and culturing, namely taking engineering bacteria seeds containing plasmids from a seed bank, inoculating the engineering bacteria seeds to an antibiotic screening culture medium, and amplifying and culturing in a fermentation tank to make the plasmids largely replicated to obtain a culture;

② Performing crude extraction, namely centrifuging a culture in a fermentation tank to obtain bacterial sludge, sequentially adding heavy suspension into the bacterial sludge, mixing, standing, removing floating impurities, filtering, and performing ultrafiltration concentration to obtain crude extract plasmid DNA;

③ Purifying the crude extract plasmid DNA to obtain supercoiled plasmid;

④ Linearization, namely cutting the supercoiled plasmid by using restriction endonuclease, and purifying by anion chromatography to obtain a linearization plasmid template;

s3, preparing mRNA stock solution:

① Synthesizing mRNA, mixing linearization plasmid DNA template, RNA polymerase and NTPs to synthesize mRNA;

② Purifying, namely purifying the synthesized mRNA to obtain mRNA stock solution;

encapsulation of mrna stock:

① Obtaining nano particles, diluting mRNA stock solution to be used as a water phase, mixing the water phase and an organic phase according to the proportion, and preparing the nano particles by a microfluidic preparation instrument;

② Solidifying and purifying, adding PBS buffer solution into the nano particles to solidify to obtain LNP particles, purifying and filtering to obtain the LNP encapsulated pharmaceutical composition of mRNA, namely the MenB vaccine.

In one embodiment the method for preparing a prophylactic MenB vaccine comprises the steps of:

S1, construction of a recombinant strain:

I. Construction of recombinant plasmids:

① According to the optimized self-replicating mRNA sequences of the example 1, the example 2, the example 3, the example 4, the example 5 and the example 6 in the second group of experiments, after sequencing verification, salI+XbaI double digestion is carried out on the 4 antigen-linked genes with SalI carried on the upstream and XbaI carried on the downstream and the self-replicating vector JJ-Sap-2;

② And (3) recovering the 4 antigen connecting genes and the self-replicating vector after enzyme digestion, preparing a reaction system according to the molar molecular ratio of the connecting genes to the vector of which the dosage ratio is 3-5:1, adding T4DNA ligase and a reaction buffer solution, uniformly mixing, and connecting at room temperature for 30-60min or connecting overnight at 16 ℃ to obtain the recombinant plasmid.

II, obtaining recombinant strains:

① Adding 3-5 mu L of the connection product of the recombinant plasmid into 50 mu L of thawed competent cells DH5 alpha, uniformly mixing, and performing heat shock, wherein the heat shock temperature is 42 ℃, and the heat shock time is 30-60s;

② The E.coli cells after heat shock were added to 500. Mu.L of liquid LB medium, cultured in a constant temperature shaking incubator at 37℃and 200rpm for about one hour, and then spread on solid LB medium, and cultured in a constant temperature incubator at 37℃for 16 to 18 hours.

S2, preparing recombinant plasmids:

① Selecting single bacterial colony with good growth vigor, adding the single bacterial colony into a liquid synthetic culture medium, culturing the single bacterial colony in a 200rpm constant-temperature shaking incubator for 4-6 hours at 37 ℃, extracting plasmids from a small amount of bacterial liquid by using a plasmid extraction kit, and then carrying out SalI+XbaI enzyme digestion identification;

② Amplifying and culturing the bacterial liquid with correct enzyme cutting identification result in a constant-temperature shaking incubator at 37 ℃ and 200rpm for 14-16 hours, centrifuging at 4000rpm for about 5-10min, and obtaining bacterial sludge;

③ The collected bacterial sludge is cracked, the lysate I is added, the bacteria are fully suspended by using an oscillator, the volume ratio of the bacterial sludge to the lysate I is 4-6:250, after the bacterial is fully suspended, the lysate II is added, the bacteria are cracked by gently reversing and uniformly mixing, the lysate III is added, the bacteria are immediately and gently reversing and uniformly mixing, the bacteria are fully neutralized, and the bacteria are placed at room temperature for 5-10min. The method comprises the steps of adding a cleaning solution into a DNA adsorption column, adding a cleaning solution into the adsorption column, centrifuging at 12000rpm for 1min to remove the waste liquid, adding an eluent, standing at 37 ℃ for 2-5min, centrifuging at 12000rpm for 1min, and collecting the eluent to obtain the annular plasmid DNA solution, wherein the volume ratio of the lysate I to the lysate III is 5:5:7, the volume ratio of the lysate III to the lysate 12000rpm is 5-10min, and the supernatant is added into the DNA adsorption column and centrifuged at 12000rpm for 1min to remove the waste liquid.

S3, preparing mRNA stock solution:

I. Plasmid linearization:

① Using BspQ enzyme cutting system, incubating for 1-2 hours at 50 ℃, cutting the circular plasmid into linearization plasmid, taking a part of enzyme cutting product to carry out agarose gel electrophoresis verification;

② And adding 0.5 times of magnetic beads into the rest enzyme-digested product, and fully and uniformly mixing. Incubating for 10-15min at room temperature, placing on a magnetic rack, and removing supernatant after the solution is clarified;

③ The magnetic beads are rinsed by a freshly prepared 80% ethanol solution, the supernatant is removed, the magnetic beads are dried, a proper volume of Nuclear-freeH ₂ O is added for blowing and evenly mixing, and the supernatant is sucked to obtain a purified linear plasmid DNA solution and stored at-20 ℃.

II in vitro transcription reaction IVT (InVitroTranscription)

① Mixing substrates such as a linear plasmid DNA template, NTPs, cleanCapAU, a buffer solution and the like, adding T7RNA polymerase, uniformly mixing, placing in a 37-DEG CPCR instrument for in-vitro transcription reaction, and adding DNaseI to terminate the reaction after 1-3 hours of reaction;

② Adding Nuclease-free water and LiCl solution into the reacted solution, wherein the volume ratio of the reacted solution to the water and LiCl is 1:1.5:1.5, incubating for 15-30min at-20 ℃ after reversing and mixing uniformly, centrifuging for 10-15min at 4 ℃ and 12000g to remove waste liquid, adding 70% ethanol to clean impurities, adding Nuclease-free water after removing supernatant, dissolving precipitate to obtain mRNA stock solution, and storing at-80 ℃.

S4. Encapsulation of mRNA stock solution

① Thawing the stock mRNA in a cold water bath, and diluting the stock mRNA to a concentration of about 0.1-0.5mg/ml by using a citrate buffer solution to obtain an mRNA working solution;

② The self-replicating mRNA is wrapped in LNP, the molar concentration ratio of cations in the lipid phase to DSPC, CHO-HP and DMP-PEG2000 is (30-60): (5-10): (30-60): (2-5), the total molar concentration of the lipid phase is 15-20mmol/L, and the cations in the lipid phase comprise DLin-MC3-DMA, JK-0042, JK-0043 and JK-0045.

③ The volume ratio of the organic phase to the mRNA working solution is 1 (2-4), and nano-particle LNP is prepared by a microfluidic preparation instrument;

④ Adding PBS buffer solution with the volume of 5-10 times of the volume of the obtained nano particles for dilution, using an ultrafiltration centrifuge tube with 100KD for centrifugal concentration at 4 ℃ and 1500g, adding PBS buffer solution with the volume of 5-10 times for dilution when the concentration is about the volume of the LNP obtained initially, continuing to concentrate to the volume of the expected concentration, and filtering by using a 0.2 mu m membrane to obtain an LNP finished product, thereby obtaining the vaccine for preventing MenB.

The prophylactic MenB vaccine was stored at-20 ℃.

S1, constructing a vector, namely cloning a self-replication mRNA sequence to the self-replication vector through SalI/XbaI double enzyme digestion to construct a self-replication mRNA plasmid, and then introducing the DNA sequence of the self-replication mRNA plasmid into an escherichia coli cell by adopting a thermal shock method to establish a research and development seed library;

S2, plasmid preparation:

① And (3) taking engineering bacteria seeds in the research and development seed library, adding the engineering bacteria seeds into a liquid synthetic culture medium shaking incubator, and detecting that the OD600 of the seeds reaches 0.6-2.5. Amplifying and culturing the amplified seeds in a fermentation tank for 38-45 hours, controlling the fermentation temperature to be 28-37 ℃, controlling the rotation speed to be 100-1000 rpm, associating the dissolved oxygen with the stirring rotation speed, controlling the pH to be 6.3-7.0, and supplementing the pH, centrifuging at 4000rpm for 5min after fermentation is finished, and removing supernatant to obtain bacterial mud;

② Mixing the bacterial mud with the heavy suspension S1 in a mass ratio of 1:5-15. Adding 1-3 times of S2 solution, standing for 2-10 min, adding 1-5 times of S3 solution and ammonium bicarbonate, standing for 30-60min, removing floating impurities, filtering with a filter, concentrating the solution with an ultrafiltration system, wherein the pH value of the S1 solution is 7.5-8.5, the solution consists of 25mM-100mM glucose, 15mM-35mMTri-HCl and 5mM-20mM EDTA, the S2 solution consists of 0.1M-0.3MNaOH and 0.5% -2% SDS, and the S3 solution consists of a mixed solution formed by 3MKAc and 2 MHAc.

③ The molecular sieve chromatographic column is used for chromatography, the linear flow rate of the chromatography is 1-4 cm/h, the target product flows through the place of about 1/3CV after the beginning of the sample loading, the UV curve rises, and the flow through peak is collected. The buffer solution used for the chromatography of the molecular sieve chromatographic column consists of 1.8M-2.3M (NH ₄)₂SO₄, 0.2M-0.5MNaCl, 0.1MTris-HCl and 0.01MEDTA-2 Na).

④ Chromatography was performed using an affinity column, equilibrated with buffer A, and the linear flow rate of chromatography was 10-15 cm/hr. After loading, 5% -15% buffer solution B is used for cleaning impurities, and the cleaning speed is about 2-5CV.

The buffer solution A consists of 1.8M-2.3M (NH ₄)₂SO₄, 0.1M Tris-HCl,0.01MEDTA-2 Na;

The buffer solution B consists of 1.8M-2.3M (NH ₄)₂SO₄, 0.2M-0.5M NaCl,0.1M Tris-HCl,0.01M EDTA-2 Na) and is eluted with 20% -50% buffer solution B, and the elution peak is collected.

⑤ And (3) changing the solution by using an ultrafiltration system, changing the salt solution into a1 xTE buffer solution, and selecting 100kD-500kD from the hollow fiber column.

⑥ Mixing supercoiled plasmid DNA, linearization enzyme and water, incubating at 45-55 ℃ for 0.5-4 hours, purifying by using anion chromatographic column chromatography and ultrafiltration to obtain a linearized plasmid DNA template, and preserving at-80 ℃, wherein the chromatographic linear flow rate is 10-15 cm/hour, the chromatographic 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 the chromatographic solution A is 7.5;

the chromatographic solution B consists of EDTA-2Na and Tris-HCl, wherein the mass ratio of the EDTA-2Na to the Tris-HCl is 1.681:7.88, and the pH of the chromatographic solution B is 7.5.

S3, preparing mRNA stock solution:

① Mixing a linearized plasmid DNA template, NTPs and a buffer substrate, adding polymerase, and reacting for 1-3 hours at 33-37 ℃ in a synthesis workstation;

② Purifying by affinity chromatographic column chromatography, selecting OligodT as chromatographic packing, diluting the reaction solution 15-30 times with equilibration solution before purifying, and washing impurities for 2-8CV at linear flow rate of 6-20 cm/hr. The balance liquid consists of NaCl, EDTA and TrisHCl, and the mass ratio of the balance liquid to the EDTA is 9.35:0.067:0.32, and the elution peak is collected by pure water elution after the impurity washing is finished;

③ Purification was performed using a TFF system with pure water as the displacement solution. The transmembrane pressure is 5-20psi, the shearing rate is 1000-4000/sec, the washing and filtering are 8-15 times, and the concentration is carried out again to the target concentration. The obtained sample was filtered with 0.2 μm to obtain mRNA stock solution, which was stored at-80 ℃.

Encapsulation of mrna stock:

① Thawing the stock mRNA in cold water bath, diluting the stock mRNA to 0.1-0.5 mg/ml with buffer salt, and labeling to obtain RNA working solution.

The volume ratio of the organic phase to the aqueous phase RNA working solution is 1:3, and the nano particles are prepared by a microfluidic preparation instrument.

② The LNP particles were solidified by adding 1 XPBS, which is 4-20 times the volume of the harvested nanoparticles, and purified by using a TFF ultrafiltration system, with 1 XPBS as the displacement solution. The transmembrane pressure is 10psi, the shear rate is 2000/sec, the dilution concentration is 4-8 times, the washing filtration is 4-8 cycles, and the concentration is 2-3 times. The obtained sample was filtered with 0.2 μm to obtain an LNP-encapsulated pharmaceutical composition of mRNA, and a prophylactic MenB vaccine was obtained and stored at-20 ℃.

The inventor finds that the preparation method of the MenB vaccine can be used for obtaining the MenB vaccine, so that the vaccine has the advantages of low injection needle number, low dosage and high expression effect, and the possible reason is that the SARNA can be self-replicated in cells to serve as a carrier, so that the dosage and frequency can be reduced, and meanwhile, the expression efficiency of the vaccine is improved, so that a better immune effect is expected to be achieved.

The inventor finds that the preparation method of the preventive MenB vaccine can be used for obtaining the preventive MenB vaccine in different preparation forms in the experimental process, so that the drug effect of the vaccine can be improved, and the preventive MenB vaccine in different preparation forms can have different effects on the drug effect in the drug effect experiment, and the vaccine after technological improvement has the advantages of fast immune response initiation, high antibody level and long-term immune protection.

Drawings

FIG. 1 shows the result of an electrophoresis test on a self-replicating mRNA sequence SEQ ID NO. 15 and a recombinant plasmid thereof.

FIG. 2 shows the result of an electrophoresis test on a self-replicating mRNA sequence SEQ ID NO. 17 and a recombinant plasmid thereof.

FIG. 3 shows the results of an electrophoresis test on the self-replicating mRNA sequence SEQ ID NO. 19 and a recombinant plasmid thereof.

FIG. 4 shows the results of electrophoresis tests performed on the self-replicating mRNA sequence SEQ ID NO. 21 and recombinant plasmids thereof.

FIG. 5 shows the results of an electrophoresis test on the self-replicating mRNA sequence SEQ ID NO. 23 and recombinant plasmid thereof.

FIG. 6 shows the result of an electrophoresis test on a self-replicating mRNA sequence SEQ ID NO. 25 and a recombinant plasmid thereof.

FIG. 7 shows the results of protein bands detected by immunoblotting of the sequences provided in examples 7, 8, 9, 10, 11 and 12.

FIG. 8 is a graph showing the detection results of fHbp-binding antibodies.

FIG. 9 is a graph showing the detection results of NHBA binding antibodies.

FIG. 10 is a graph showing the detection results of NadA binding antibodies.

FIG. 11 is a graph of the detection results of porA binding antibodies.

FIG. 12 is a graph showing the results of FACs detection after D34 days of the injection of the preventive MenB vaccine.

FIG. 13 is a graph showing the results of FACs detection after D70 days of the injection of the preventive MenB vaccine.

FIG. 14 is a graph showing the results of ELISPOT assay on day 34 of injection of the preventive MenB vaccine.

FIG. 15 is a graph showing the results of ELISPOT assay on day 70 of injection of the preventive MenB vaccine.

FIG. 16 shows the results of the MenB (PorA) binding antibody titer assay.

FIG. 17 shows the results of measurement of the titer of MenB (NadA) binding antibodies.

FIG. 18 shows the results of the MenB (NHBA) binding antibody titer assay.

FIG. 19 shows the results of measurement of the titre of MenB (fHbp) binding antibodies.

FIG. 20 shows the results of CD4+ IFNg+ T cell typing assays.

FIG. 21 shows the results of CD4+CD69+ T cell typing assays.

FIG. 22 shows the results of CD8+IFNg+T cell typing assays.

FIG. 23 shows the results of CD8+CD69+ T cell typing assays.

FIG. 24 shows ELISPOT assay results at day D35 of injection.

FIG. 25 shows ELISPOT assay results at day 70 of D injection.

FIG. 26 shows the results of serum bactericidal titres of the SEQIDNO 17 resolution-preparation mixtures.

FIG. 27 shows the results of serum bactericidal titres of the SEQIDNO 15 resolution-preparation mixtures.

FIG. 16 a shows the results of serum-binding antibody detection with respect to PorA antigen protein when the amount 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-resolution-stock solution mixture provided in example 15, and the resolution-preparation mixture provided in example 16 were 1. Mu.g.

FIG. 16b shows the results of serum-binding antibody detection with respect to PorA antigen protein when the amount of the self-replicating mRNA sequence SEQ ID NO:15 as provided in example 1, the self-replicating mRNA sequence SEQ ID NO:17 as provided in example 2, the SEQ ID NO: 17-split-stock mixture as provided in example 15, and the split-preparation mixture as provided in example 16 were 5. Mu.g.

FIG. 16 c shows the results of serum-binding antibody detection with respect to PorA antigen protein when the amount of the self-replicating mRNA sequence SEQ ID NO:15 as provided in example 1, the self-replicating mRNA sequence SEQ ID NO:17 as provided in example 2, the SEQ ID NO: 17-split-stock mixture as provided in example 15, and the split-preparation mixture as provided in example 16 were 15. Mu.g.

FIG. 17 a shows the results of serum-binding antibody detection of binding NadA antigen protein when the amount of the self-replicating mRNA sequence SEQ ID NO:15 as provided in example 1, the self-replicating mRNA sequence SEQ ID NO:17 as provided in example 2, the SEQ ID NO: 17-split-stock solution mixture as provided in example 15 and the split-preparation mixture as provided in example 16 was 1. Mu.g.

FIG. 17 b shows the results of serum-binding antibody detection with NadA antigen protein when the amount of the self-replicating mRNA sequence SEQ ID NO:15 as provided in example 1, the self-replicating mRNA sequence SEQ ID NO:17 as provided in example 2, the SEQ ID NO: 17-split-stock mixture as provided in example 15, and the split-preparation mixture as provided in example 16 were 5. Mu.g.

FIG. 17 c shows the results of serum-binding antibody detection with NadA antigen protein when the amount of the self-replicating mRNA sequence SEQ ID NO:15 as provided in example 1, the self-replicating mRNA sequence SEQ ID NO:17 as provided in example 2, the SEQ ID NO: 17-split-stock mixture as provided in example 15, and the split-preparation mixture as provided in example 16 were 15. Mu.g.

FIG. 18 a shows the results of serum-binding antibody detection of binding NHBA antigen protein when the amount of the self-replicating mRNA sequence SEQ ID NO:15 as provided in example 1, the self-replicating mRNA sequence SEQ ID NO:17 as provided in example 2, the SEQ ID NO: 17-split-stock mixture as provided in example 15 and the split-preparation mixture as provided in example 16 was 1. Mu.g.

FIG. 18 b shows the results of serum-binding antibody detection of binding NHBA antigen protein when the amount of the self-replicating mRNA sequence SEQ ID NO:15 as provided in example 1, the self-replicating mRNA sequence SEQ ID NO:17 as provided in example 2, the SEQ ID NO: 17-split-stock mixture as provided in example 15 and the split-preparation mixture as provided in example 16 was 5. Mu.g.

FIG. 18 c shows the results of serum-binding antibody detection of binding NHBA antigen protein at 15. Mu.g 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 mixture provided in example 15 and the split-preparation mixture provided in example 16.

FIG. 19 a shows the results of serum-binding antibody detection of binding to fHbp antigen protein at1 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 mixture provided in example 15, and the split-preparation mixture provided in example 16.

FIG. 19 b shows the results of serum-binding antibody detection of binding to fHbp antigen protein at 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 mixture provided in example 15, and the split-preparation mixture provided in example 16.

FIG. 19 c shows the results of serum-binding antibody detection of binding to fHbp antigen protein at 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 mixture provided in example 15, and the split-preparation mixture provided in example 16.

FIG. 24 a shows the results of ELISPOT assay for D35 days when 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 were used in an amount of 1. Mu.g.

FIG. 24 b shows the results of ELISPOT assay for D35 days when 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 were used in an amount of 5. Mu.g.

FIG. 24 c shows the results of ELISPOT assay for D35 days when 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 were used in an amount of 5. Mu.g.

FIG. 25 a shows the results of ELISPOT assay for D70 days when 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 were used in an amount of 1. Mu.g.

FIG. 25 b shows the results of ELISPOT assay for D70 days when 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 were used in an amount of 5. Mu.g.

FIG. 25 c shows the results of ELISPOT assay for D70 days when 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 were used in an amount of 5. Mu.g.

Advantageous effects

1. The application provides a MenB vaccine for preventing and reducing the death rate of domestic infection caused by B-type meningococcus aiming at the strain screening amino acid sequence of which the coverage is found in Chinese monitoring.

2. According to the application, for the amino acid which can be mutated, MITD molecular sequences are added at the tail end of the serial sequences, so that the antigen can be guided to enter a specific region of the dendritic cells, and the antigen is easier to be treated by the dendritic cells and presented to the T cells, thereby starting the immune response.

3. In the application, a signal peptide sequence is added at the 5' end of the tandem sequence to guide the new-born protein into a specific secretion path in cells, ensure that the protein can be correctly positioned and play a role, ensure the secretion of self-replicase and antigen, and discover that different signal peptides can have different guiding characteristics in the experimental process, thereby being applicable to different cellular environments and protein functional requirements.

4. The present application provides for precise control of protein translation by adding a stop codon to terminate optimized protein synthesis to produce a desired protein variant or to avoid the production of unwanted proteins.

5. The tandem sequence provided by the application can ensure that protein domains coded by individual sequences in the tandem sequence can independently play a role, avoid steric hindrance and interference between the individual sequences, limit the tandem sequence to be connected between the individual sequences by using a flexible Linker as shown in SEQ ID NO:11, and can provide enough space freedom for each protein domain to enable the protein domain to freely fold and move, thereby ensuring the correct conformation and functional integrity of the whole tandem protein.

6. The application can make self replication in cells as a carrier through the SARNA, so that the anti-MenB vaccine enables the vaccine to have low injection needle number, low dosage and high expression effect, the dosage and frequency can be reduced, and the expression efficiency of the vaccine can be improved, thereby achieving better immune effect.

Detailed Description

For a better understanding of the present invention, embodiments are described in detail below with reference to the drawings. It is to be understood that this example is for illustration of the invention only and is not intended to limit the scope of the invention.

The technical means adopted in this embodiment are conventional technical means in the art unless specifically described otherwise.

First set of experiments

The first set of experiments screened amino acid sequences.

For strains found in China monitoring of coverage, the protein sequences of the application cover the ST-4821, ST-41/44, ST-32, ST-198, ST-175 clone complexes in Neisseria meningitidis serogroup B bacteria. Screening an amino acid sequence aiming at Chinese strains, optimizing and deleting original signal peptide sequences aiming at fHbp and NHBA, nadA, porA antigen proteins, wherein the amino acid sequence is SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 or SEQ ID NO. 10.

The fHbp antigen protein molecule deleter 1-19 signal peptide sequence is used as an amino acid sequence SEQ ID NO. 7.

The NHBA antigen protein molecule deleter 1-17 signal peptide sequence is used as an amino acid sequence SEQ ID NO. 8.

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

The PorA antigen protein molecule deleter 1-18 signal peptide sequence is used as an amino acid sequence SEQ ID NO. 10.

Second set of experiments

The second set of experiments provided self-replicating mRNA sequences, designed in tandem for fHbp, NHBA, nadA, porA antigen proteins as self-replicating mRNA sequences.

The second set of experiments included example 1, example 2, example 3, example 4, example 5, example 6.

The self-replicating mRNA sequence 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.

The self-replicating mRNA sequence provided in example 6 is SEQ ID NO. 25.

The self-replicating mRNA sequence comprises RNA of a coding region in an amino acid sequence provided by a first group of experiments, and the amino acid sequence is formed by tandem connection of single sequences shown by SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 or SEQ ID NO. 10 according to any sequence.

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

The tandem sequences were linked between the individual sequences using a flexible Linker as shown in SEQ ID NO. 11.

And a MITD molecular sequence designed by mutation is added at the tail end of the series sequence, and the MITD molecular sequence is an amino acid sequence SEQ ID NO. 13.

Example 1

Based on a second set of experiments, a self-replicating mRNA sequence is provided, and the embodiment carries out molecular tandem design on four antigen proteins of fHbp, NHBA, nadA, porA to obtain novel coronavirus SP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA target protein amino acid, wherein the amino acid sequence is SEQ ID NO:14.

In the embodiment, the GC content and the secondary structure of the target gene SEQ ID NO. 14 are optimized by adjusting the codon sequence of the target gene SEQ ID NO. 15, so that the optimized base sequence SEQ ID NO. 15 is obtained, and the self-replicating mRNA sequence is obtained.

Example 2

Based on a second set of experiments, a self-replicating mRNA sequence is provided, and the embodiment carries out molecular tandem design on four antigen proteins of fHbp, NHBA, nadA, porA to obtain tPASP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA target protein amino acid, wherein the target protein amino acid sequence is SEQ ID NO:16.

In the embodiment, the GC content and the secondary structure of the target gene SEQ ID NO. 16 are optimized by adjusting the codon sequence of the target gene SEQ ID NO. 17, so that the optimized base sequence SEQ ID NO. 17 is obtained, and the self-replicating mRNA sequence is obtained.

Example 3

Based on a second set of experiments, a self-replicating mRNA sequence is provided, and the embodiment carries out molecular tandem design on four antigen proteins of fHbp, NHBA, nadA, porA to obtain fHbpSP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA target protein amino acid, wherein the target protein amino acid sequence is SEQ ID NO:18.

In the embodiment, the GC content and the secondary structure of the target gene SEQ ID NO. 18 are optimized by adjusting the codon sequence of the target gene SEQ ID NO. 19, so that the optimized base sequence SEQ ID NO. 19 is obtained, and the self-replicating mRNA sequence is obtained.

Example 4

Based on a second set of experiments, a self-replicating mRNA sequence is provided, and the embodiment carries out molecular tandem design on four antigen proteins of fHbp, NHBA, nadA, porA to obtain NHBASP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA target protein amino acid, wherein the target protein amino acid sequence is SEQ ID NO:20.

In the embodiment, the GC content and the secondary structure of the target gene SEQ ID NO. 20 are optimized by adjusting the codon sequence of the target gene SEQ ID NO. 21, so that the optimized base sequence SEQ ID NO. 21 is obtained, and the self-replicating mRNA sequence is obtained.

Example 5

Based on a second set of experiments, a self-replicating mRNA sequence is provided, and the embodiment carries out molecular tandem design on four antigen proteins of fHbp, NHBA, nadA, porA to obtain NadASP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA target protein amino acid, wherein the target protein amino acid sequence is SEQ ID NO:22.

In the embodiment, the GC content and the secondary structure of the target gene SEQ ID NO. 22 are optimized by adjusting the codon sequence of the target gene SEQ ID NO. 23, so that the optimized base sequence SEQ ID NO. 23 is obtained, and the self-replicating mRNA sequence is obtained.

Example 6

Based on a second set of experiments, a self-replicating mRNA sequence is provided, and the embodiment carries out molecular tandem design on four antigen proteins of fHbp, NHBA, nadA, porA to obtain PorASP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA target protein amino acid, wherein the target protein amino acid sequence is SEQ ID NO:24.

In the embodiment, the GC content and the secondary structure of the target gene SEQ ID NO. 24 are optimized by adjusting the codon sequence of the target gene SEQ ID NO. 25, so that the optimized base sequence SEQ ID NO. 25 is obtained, and the self-replicating mRNA sequence is obtained.

The second set of experiments provided a self-replicating mRNA sequence that was optimized using SEQ ID NO:26 as a stop codon.

The self-replicating mRNA sequence comprises an amino acid sequence shown as SEQ ID NO. 17.

Third set of experiments

The third set of experiments provides a prophylactic MenB vaccine and a method of preparing the same.

The prophylactic MenB vaccine included self-replicating mRNA sequences provided in the second set of experiments.

The third set of experiments included examples 7,8, 9, 10, 11, 12.

The self-replicating mRNA sequence adopted in the MenB vaccine is SEQ ID NO. 15.

The self-replicating mRNA sequence adopted in the MenB vaccine for prevention provided in the example 8 is SEQ ID NO. 17.

The self-replicating mRNA sequence adopted in the MenB vaccine for prevention provided in the example 9 is SEQ ID NO. 19.

The self-replicating mRNA sequence adopted in the MenB vaccine is SEQ ID NO. 21.

The self-replicating mRNA sequence adopted in the MenB vaccine is SEQ ID NO. 23.

The self-replicating mRNA sequence adopted in the MenB vaccine for prevention provided in the example 12 is SEQ ID NO. 25.

The preparation methods of the preventive MenB vaccine provided in the examples 7, 8, 9, 10, 11 and 12 are prepared according to the following method steps.

The preparation method of the preventive MenB vaccine comprises the following steps:

S1, construction of a recombinant strain:

I. Construction of recombinant plasmids:

② And (3) recovering the 4 antigen connecting genes and the self-replicating vector after enzyme digestion, preparing a reaction system according to the molar ratio of the connecting genes to the vector of 5:1, adding the T4DNA ligase and the reaction buffer solution, uniformly mixing, and connecting at room temperature for 30min to obtain the recombinant plasmid.

II, obtaining recombinant strains:

① Adding 5 mu L of the connection product of the recombinant plasmid into 50 mu L of thawed competent cells DH5 alpha, uniformly mixing, and carrying out heat shock, wherein the heat shock temperature is 42 ℃, and the heat shock time is 60s;

② The E.coli cells after heat shock were added to 500. Mu.L of the liquid LB medium, cultured in a constant temperature shaking incubator at 37℃and 200rpm for about one hour, and then spread on the solid LB medium, and cultured in the constant temperature incubator at 37℃for 16 hours.

S2, preparing recombinant plasmids:

① Selecting single bacterial colony with good growth vigor, adding the single bacterial colony into a liquid synthetic culture medium, culturing the single bacterial colony for 4 hours in a 200rpm constant-temperature shaking incubator at 37 ℃, extracting plasmids from a small amount of bacterial liquid by using a plasmid extraction kit, and then carrying out SalI+XbaI enzyme digestion identification;

② Amplifying and culturing the bacterial liquid with correct enzyme cutting identification result in a constant-temperature shaking incubator at 37 ℃ and 200rpm for 14 hours, and centrifuging at 4000rpm for about 10 minutes to obtain bacterial sludge;

③ The collected bacterial sludge is cracked, the lysate I is added, the bacteria are fully suspended by using an oscillator, the volume ratio of the bacterial sludge to the lysate I is 4:250, after the bacterial is fully suspended, the lysate II is added, the bacteria are cracked by gently reversing and uniformly mixing, the lysate III is added, the lysate III is immediately and gently reversing and uniformly mixing, the bacteria are fully neutralized, and the bacterial sludge and the lysate I are placed at room temperature for 5min. The volume ratio of the lysate I to the lysate II to the lysate III is 5:5:7, the mixture after the lysis is centrifuged at 12000rpm for 5min, the supernatant is taken and added into a DNA adsorption column, the supernatant is centrifuged at 12000rpm for 1min to remove waste liquid, a cleaning solution is added into the adsorption column, the supernatant is centrifuged at 12000rpm for 1min to remove waste liquid, an eluent is added, the mixture is kept stand at 37 ℃ for 5min, and the eluent is collected to obtain the DNA solution of the annular plasmid.

S3, preparing mRNA stock solution:

I. Plasmid linearization:

① Using BspQ to 1 enzyme cutting system, incubating for 1 hour at 50 ℃, cutting the circular plasmid into linearization plasmid, taking 20 mug enzyme cutting product to carry out agarose gel electrophoresis verification;

② Adding 1.0 times of enzyme cutting product into the reaction system, adding 0.5 times of magnetic beads, and fully and uniformly mixing. Incubating for 10min at room temperature, placing on a magnetic rack, and removing the supernatant after the solution is clarified;

③ The magnetic beads are rinsed by a freshly prepared 80% ethanol solution, the supernatant is removed, the magnetic beads are dried, and then a 0.1-time reaction system, namely, nuclear-freeH ₂ O without enzyme water, is added for blowing and uniformly mixing, and the supernatant is sucked to obtain a purified linear plasmid DNA solution and is stored at the temperature of minus 20 ℃.

The enzyme digestion product is XbaI enzyme, and the brand of the purchasing manufacturer is the company of Shanghai Biotechnology (Shanghai) stock.

II in vitro transcription reaction IVT (InVitroTranscription)

① Mixing a linear plasmid DNA template, NTPs, cleanCapAU and Tris-HCl buffer solution as substrates, adding T7RNA polymerase, uniformly mixing, placing in a 37 DEG CPCR instrument for in-vitro transcription reaction, and adding DNaseI for termination reaction after 3 hours, wherein the ratio of the mass of the DNA template to the DNaseI volume is 1g to 3 mu L.

② Adding Nuclear-free water and LiCl solution into the reacted solution, wherein the volume ratio of the reacted solution to the water and LiCl is 1:1.5:1.5, incubating for 15min at-20 ℃ after reversing and mixing uniformly, centrifuging for 10min at 4 ℃ and 12000g to remove waste liquid, adding 70% ethanol to clean impurities, adding Nuclear-free water after removing supernatant, dissolving precipitate to obtain mRNA stock solution, and storing at-80 ℃.

The concentration of the DNA template is 0.05-0.1 mug/mu L.

The NTPs are used in an amount of 0.1 times the total volume.

The CleanCapAU is used in an amount of 0.1 times the total volume.

The amount of the Tris-HCl buffer solution is 0.1 times of the total volume.

The amount of T7RNA polymerase was 0.5 times the total volume.

The DNA template, NTPs, cleanCapAU, tris-HCl buffer, T7RNA polymerase and DNaseI are all commercial products.

The manufacturer brand of the DNA template may be Nanjinouzan Biotechnology Co., ltd.

The manufacturer brand of the NTPs may be the biological technology division of nanking and nuozhenz.

The manufacturer brand of CleanCapAU may be Jiangsu Shenji Biotechnology Co.

The manufacturer brand of the Tris-HCl buffer can be the Simer Feishmania technology company.

The manufacturer of the T7RNA polymerase may be sold under the brand name Simer Feishmania technology.

The manufacturer brand of dnaseli may be sameir femto technology.

S4. Encapsulation of mRNA stock solution

① Thawing the stock mRNA in a cold water bath, and diluting the stock mRNA to a concentration of about 0.5mg/ml by using a citrate buffer solution to obtain an mRNA working solution, wherein the pH value of the citrate buffer solution is 5.0.

② Wrapping the self-replicating mRNA in LNP to obtain an organic phase (lipid phase) with a molar concentration ratio of cations to DSPC, CHO-HP and DMP-PEG2000 of 30:5:30:2, the total molar concentration of the lipid phase being 15mmol/L;

③ The volume ratio of the organic phase to the mRNA working solution is 1:2, and the nano-particle LNP is prepared by a microfluidic preparation instrument;

④ And adding PBS buffer solution with the volume of 5 times of the volume of the obtained nano particles for dilution, using an ultrafiltration centrifuge tube with the volume of 100KD for centrifugal concentration at the temperature of 4 ℃ and under the condition of 1500g, adding PBS buffer solution with the volume of 5 times for dilution when the concentration is about the volume of LNP obtained initially, continuously concentrating to 1-0.5 time of the volume before dilution, and filtering by using a 0.2 mu m membrane to obtain an LNP finished product, thereby obtaining the vaccine for preventing MenB.

The cationic model in the lipid phase can be any one of DLin-MC3-DMA, JK-0042, JK-0043 and JK-0045, and the cationic model in the lipid phase adopted in the experiment is JK-0042.

The prophylactic MenB vaccine was stored at-20 ℃.

The pH of the PBS buffer was 7.5.

The manufacturer of the DLin-MC3-DMA is Xiamen Saunobang Biotechnology Co., ltd.

The JK-0042 is derived from Ningbo Jun health biotechnology Co.

The JK-0043 is derived from Ningbo Jun health biotechnology Co.

The JK-0045 is derived from Ningbo Jun health biotechnology Co.

Fourth set of experiments

The fourth set of experiments included examples 13, 14, 15, 16.

In example 13, four antigen proteins fHbp, NHBA, nadA, porA of seq id no:15 sequence 1 in the self-replicating mRNA sequence were designed separately and mixed in stock prior to encapsulation to prepare a formulation, defined as seq id no: 15-split-stock mixture.

In example 14, four antigen proteins of fHbp, NHBA, nadA, porA of seq id no:15 sequence 1 in the self-replicating mRNA sequence were designed separately and mixed after encapsulation, followed by preparation purification and split charging, defined as seq id no: 15-split-preparation mixture.

In example 15, four antigen proteins fHbp, NHBA, nadA, porA of seq id no:17 sequence 1 in the self-replicating mRNA sequence were designed separately and mixed in stock prior to encapsulation to prepare a formulation, defined as seq id no: 17-split-stock mixture.

In example 16, four antigen proteins, fHbp, NHBA, nadA, porA of seq id no:17 sequence 1 in the self-replicating mRNA sequence were designed separately and mixed after encapsulation, followed by preparation purification and split charging, defined as seq id no: 17-split-preparation mixture.

The encapsulation methods employed in examples 13, 14, 15 and 16 include the following steps:

S2, plasmid preparation:

① And (3) taking engineering bacteria seeds in the research and development seed library, adding the engineering bacteria seeds into a liquid synthetic culture medium shaking incubator, and detecting that the OD600 of the seeds reaches 0.6-2.5. Amplifying and culturing the amplified seeds in a fermentation tank for 40 hours, controlling the fermentation temperature at 30 ℃, controlling the rotation speed at 500rpm, controlling the pH at 6.3-7.0, and controlling the feeding pH, centrifuging at 4000rpm for about 5min after fermentation, and removing supernatant to obtain bacterial sludge;

② And mixing the bacterial mud with the heavy suspension S1, wherein the mass ratio is 1:10. Adding 2 times of S2 solution, standing for 5 min, adding 3 times of S3 solution and ammonium bicarbonate, standing for 60min, removing floating impurities, filtering with a filter, concentrating the solution with an ultrafiltration system, wherein the pH value of the S1 solution is 7.5-8.5, the solution consists of 50mM glucose, 30mMTri-HCl and 15mM EDTA, the S2 solution consists of 0.2MNaOH and 1% SDS, and the S3 solution consists of a mixed solution formed by 3MKAc and 2 MHAc.

③ Chromatography was performed using molecular sieve column chromatography at a linear flow rate of 3 cm/hr and at about 1/3CV after the start of loading, the target product was allowed to flow through, the UV profile was raised, and the flow through peak was collected. The buffer solution used for the chromatography of the molecular sieve chromatographic column consists of 2.0M (NH 4) 2SO4, 0.5MNaCl, 0.1MTris-HCl and 0.01MEDTA-2 Na.

④ Chromatography was performed using an affinity column, equilibrated with buffer a, and the linear flow rate of chromatography was 10 cm/hr. After loading was completed, the impurities were washed with 15% buffer solution B, about 5CV.

The buffer solution A consisted of 2.3M (NH ₄)₂SO₄, 0.1M Tris-HCl,0.01MEDTA-2 Na;

The buffer solution B consisted of 2.3M (NH ₄)₂SO₄, 0.2M NaCl,0.1M Tris-HCl,0.01M EDTA-2 Na; and was eluted with 30% buffer solution B, and the elution peaks were collected.

⑤ The ultrafiltration system was used to change the saline solution to 1×TE buffer and the hollow fiber column was selected to be 300kD3.

⑥ Mixing supercoiled plasmid DNA, linearization enzyme and water, incubating for 2 hours at 50 ℃, purifying by using anion chromatographic column chromatography and ultrafiltration to obtain a linearized plasmid DNA template, and storing the linearized plasmid DNA template at-80 ℃, wherein the linear flow rate of chromatography is 10-15 cm/hour, chromatographic 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 the chromatographic solution A is 7.5;

S3, preparing mRNA stock solution:

① Taking a linearized plasmid DNA template, NTPs and Tris-HCl buffer solution as substrates, mixing, adding T7RNA polymerase, and reacting for 3 hours at 33 ℃ in a synthesis workstation;

② Purification was performed by affinity column chromatography, chromatography packing was chosen OligodT. The reaction solution was diluted 20-fold with an equilibration solution before purification, the linear flow rate for purification was 10 cm/hr, and 5CV of impurities was washed. The balance solution consists of NaCl, EDTA and TrisHCl, wherein the mass ratio of the NaCl to the EDTA to the TrisHCl is 9.35:0.067:0.32, and the elution peak is collected by pure water elution after the impurity washing is finished;

③ Purification was performed using a TFF system with pure water as the displacement solution. The transmembrane pressure was 10psi, the shear rate was 3000/sec, washed 10 times, and then concentrated to the target concentration. The obtained sample was filtered with 0.2 μm to obtain mRNA stock solution, which was stored at-80 ℃.

The concentration of the DNA template is 0.05-0.1 mug/mu L.

The NTPs are used in an amount of 0.1 times the total volume.

The amount of the Tris-HCl buffer solution is 0.1 times of the total volume.

The amount of T7RNA polymerase was 0.5 times the total volume.

The DNA template, NTPs, tris-HCl buffer solution and T7RNA polymerase are all commercial products.

Encapsulation of mrna stock:

② The self-replicating mRNA was encapsulated in LNP to obtain an organic phase (lipid phase) with a molar ratio of cations to DSPC, CHO-HP and DMP-PEG2000 of 30:5:30:2, the total molar concentration of the lipid phase being 15mmol/L.

③ The volume ratio of the organic phase to the mRNA working solution is 1:3, and the nano-particle LNP is prepared by a microfluidic preparation instrument;

④ The LNP particles were solidified by adding 10 times the volume of 1 XPBS of the harvested nanoparticles, then purified by using a TFF ultrafiltration system with a displacement solution of 1 XPBS, a transmembrane pressure of 10psi, a shear rate of 2000/sec, a dilution concentration of 6 times, a wash filtration of 6 cycles, and a re-concentration of 3 times. The obtained sample was filtered with 0.2 μm to obtain an LNP-encapsulated pharmaceutical composition of mRNA, which was stored at-20 ℃.

Performance test:

1. Electrophoresis test:

The optimized self-replicating mRNA sequences of examples 1,2, 3, 4, 5, and 6 and the recombinant plasmids obtained according to the preparation method provided in the third set of experiments were subjected to gel electrophoresis test to obtain plasmid identification patterns.

The result of the electrophoresis test for the self-replicating mRNA sequence SEQ ID NO. 15 and the recombinant plasmid thereof is shown in FIG. 1.

The result of the electrophoresis test for the self-replicating mRNA sequence SEQ ID NO:17 and the recombinant plasmid thereof is shown in FIG. 2.

The result of the electrophoresis test for the self-replicating mRNA sequence SEQ ID NO. 19 and the recombinant plasmid thereof is shown in FIG. 3.

The result of the electrophoresis test for the self-replicating mRNA sequence SEQ ID NO. 21 and the recombinant plasmid thereof is shown in FIG. 4.

The result of the electrophoresis test for the self-replicating mRNA sequence SEQ ID NO. 23 and the recombinant plasmid thereof is shown in FIG. 5.

The result of the electrophoresis test for the self-replicating mRNA sequence SEQ ID NO:25 and the recombinant plasmid thereof is shown in FIG. 6.

2. Cell experiment:

(1) Cell protein sample collection:

① HEK293 cells are cultured on a cell pore plate until the confluence of the cells of each pore reaches 80% -90%;

② Taking 2 sterile EP tubes respectively, adding 2-5 mu gmRNA samples into one tube, diluting to 150 mu L with Opti-MEM (serum-free medium), adding 3-5 mu L of transfection reagent LipofectamineTMMessengerMAXTMReagent into the other tube, supplementing 150 mu L with Opti-MEM, mixing the two tubes of liquid, blowing and mixing uniformly, standing for 5min, adding the solution after standing into an orifice plate dropwise, and culturing in a carbon dioxide incubator for 24 hours;

③ Taking out the cell pore plate, observing the cell state under a microscope, then placing on ice, collecting the culture solution in the hole, adding 1% PMSF by volume, uniformly mixing, and placing on ice;

④ Washing the cell pore plate of the sucked dry culture solution once by using ice-bath 1 XPBS, adding 200 mu LRIPA lysate into each well, adding 1% PMSF into the lysate, putting on ice for 5-10min, collecting the lysate and swirling;

⑤ Centrifuging the collected culture solution and lysate at 4 ℃ and 17000g for 15-30min, collecting supernatant to obtain a cellular protein sample, and storing at-20 ℃.

The protein bands were detected by immunoblotting using 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 and positive and blank control samples provided in examples 7, 8, 9, 10, 11 and 12, and the test results are shown in FIG. 7.

The testing method comprises the following steps:

① Adding 4X LDSsamplebuffer and 1X MDTT (10X) according to the concentration of the cellular protein sample, adding deionized water to fill up to a 60 mu l system, incubating at 90 ℃ for 5-10min, centrifuging, mixing uniformly, and loading;

② Electrophoresis, namely 80V constant-pressure electrophoresis of the laminated gel for 20-30min, wherein the concentration of the polyacrylamide separating gel is 6%, the electrophoresis condition of the separating gel is 150V constant-pressure electrophoresis for 35min, and the electrophoresis is stopped after the dye reaches the bottom of the separating gel;

③ The method comprises the steps of (1) performing electrotransfer, namely placing a PVDF film into an activating solution for activation, taking out gel, cutting off redundant parts of the gel, and manufacturing a sandwich structure in a 1X film transfer buffer solution, wherein the sponge-filter paper-gel-PVDF film-filter paper-sponge is used for guaranteeing that the sandwich structure has no bubbles, adding a proper amount of the 1X film transfer buffer solution into a film transfer tank, and placing the film transfer buffer solution into an ice box for 100min under 200mA constant current;

④ Blocking, namely preparing 5% BSA as blocking solution by using 1X TPBT, putting the transferred PVDF film into the solution, and incubating the solution on a shaking table at room temperature for 60min, and rinsing the solution for 3 times by using 1X TBST buffer solution for 10min each time;

⑤ Primary antibody incubation, namely diluting the primary antibody by using a1 Xantibody diluent according to the specification, incubating for 1 hour at room temperature after shaking, incubating overnight at 4 ℃, and recovering the room temperature after shaking for 1 hour for the next day, washing 3 times by using 1 XTBST for 5min each time;

⑥ Secondary antibody incubation, namely diluting the secondary antibody by using a1 Xantibody diluent according to the specification, incubating for 1 hour by using a 37 ℃ light-shielding shaking table, then incubating for 20min by using a room temperature shaking table, and washing for 3 times by using 1 XTBST for 5min each time;

⑦ Developing, namely uniformly mixing the developing solution A, B according to the ratio of 1:1, dripping the mixture onto a film, and placing the film into a chemiluminescent imaging system for imaging.

In FIG. 7, markers are protein molecular weight standards, including specific molecular weight markers such as 245kD and 180kD, and are used for indicating the molecular weight of proteins in samples, lane1 is a blank sample, theoretically no specific band of target proteins should be used for detecting background signals of an experimental system and eliminating interference factors such as nonspecific binding, lane2 is positive, a positive control sample contains samples with known target proteins, characteristic bands of target proteins can appear for verifying the effectiveness of the experimental system, ensuring that the experimental process can correctly detect the target proteins, lane3-Lane8 is a detection band containing related samples 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, the molecular weight of the target proteins in the samples can be judged by comparison with markers, comparison with positive control can evaluate the expression conditions of the target proteins in the samples, including whether the target proteins are successfully expressed and the relative expression level is low. The information such as the position and the gray level of the strip can provide basis for analyzing the characteristics of the target protein in each sample.

4. Animal experiments and serum binding antibody detection:

4.1 animal experiments:

① Grouping principle, wherein Balb/c mice are grouped according to a random grouping method, 5 mice are injected into each group, and the Day of grouping is Day0 after mRNA sequence preventive MenB vaccine is injected into each group.

② Animals were dosed by setting different control groups, using prepared self-replicating mRNA-LNP samples at doses of 15 μg/min, empty LNP groups as control groups, and single and double dosing at Day0 and Day21, respectively.

③ Recording test indexes, namely observing animal conditions every day and recording animal clinical symptoms. Animal clinical symptoms include, but are not limited to, 0 normal, 1 reduced activity, 2 restlessness, 3 tremors, 4 whirls or backs, 5 vertical hairs, 6 breathing abnormalities, 7 bows, 8 clear decreases in body temperature, 9 hair loss, 10 eye abnormalities, 11 abdominal swelling, 12 others.

④ The materials were obtained by retroorbital bleeding at Day0, day13, day20, day29, day34, day50, D72, and Day90, respectively.

⑤ Termination of the experiment after all dosing observations were completed, observations were continued for 1-2 months and the experiment was terminated.

4.2 Serum binding antibody detection:

The test results are shown in FIG. 8-FIG. 11, wherein the self-replicating mRNA sequence 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, which is bound to fHbp, NHBA, nadA or PorA antigen protein respectively, is subjected to serum binding antibody detection. FIG. 8 is a graph showing the detection result of fHbp binding antibody, FIG. 9 is a graph showing the detection result of NHBA binding antibody, FIG. 10 is a graph showing the detection result of nadA binding antibody, and FIG. 11 is a graph showing the detection result of porA binding antibody.

1. Mu.g, 5. Mu.g and 15. Mu.g of the self-replicating mRNA sequence SEQ ID NO:15 provided in example 1, 1. Mu.g, 5. Mu.g and 15. Mu.g of the self-replicating mRNA sequence SEQ ID NO:17 provided in example 2, 1. Mu.g, 5. Mu.g and 15. Mu.g of the SEQ ID NO: 15-split-stock solution mixture provided in example 13, 1. Mu.g, 5. Mu.g and 15. Mu.g of the split-preparation mixture provided in example 14, 1. Mu.g, 5. Mu.g and 15. Mu.g of the SEQ ID NO: 17-split-stock solution mixture provided in example 15, 1. Mu.g, 5. Mu.g and 15. Mu.g of the split-preparation mixture provided in example 16 were combined with fHbp, NHBA, nadA or PorA antigen protein, respectively, and serum binding antibody detection was performed, and the test results are shown in FIGS. 16-19.

The detection method comprises the following steps:

① Diluting MenB (fHbp) or MenB (NHBA) or MenB (NadA) or MenB (PorA) antigen to 1 μg/mL with ELISA coating buffer, adding diluted antigen into enzyme-labeled plate hole, 100 μl/hole, and coating at 4deg.C overnight (14-18 h);

② The next day, PBST is rinsed for 3 times, each time lasts for 5min, after the water is beaten, 200 mu L of 1% BSA is added into each hole to be blocked, after incubation for 1 hour at 37 ℃, PBST is rinsed for 3 times, each time lasts for 5min, and the water is beaten;

③ Adding mouse serum with the corresponding dilution ratio, making 1 compound hole for each sample, setting negative/positive control at the same time, and rinsing with PBST 3 times after incubation for 1 hour at 37 ℃ for 5min each time, and drying the water;

④ Anti-mouse IgG heavy chain antibody (HRP labeled) was added, 100. Mu.L/well and after incubation at 37℃for 1 hour, PBST was rinsed 3 times for 5min each time, and the water was drained;

⑤ Adding TMB color development liquid to develop color, incubating for 10-15min at room temperature and in dark place, and adding stop solution to stop reaction at 50 mu L/hole. Absorbance values were measured at a wavelength of 450nm using an enzyme-labeled instrument, and the titer of each bound antibody in the sample was calculated from the OD values.

As can be seen from FIG. 16, the titers of the bound antibodies of each sample group are generally lower at 14 days and 21 days after administration, and the titers of the antibodies of part of the sample groups are remarkably increased at 35 days, 49 days and 70 days after administration over time, so that the differences of different sequences, doses and the capacity and time course of inducing the production of MenB (porA) bound antibodies by the vaccine in the form of preparation are reflected, and the method can be used for evaluating the immunization effect of the vaccine and optimizing the vaccine design.

It can be seen from fig. 17 that the combined antibody titers of the majority of the sample groups were at a low level at 14 and 21 days after administration, indicating that the immune response of the body to the vaccine was not yet sufficiently initiated at this time, and that the antibody titers of the part of the sample groups began to rise significantly at 35 days after administration, indicating a gradual enhancement of the immune response of the body. 49 days and 70 days after administration, higher levels of antibody titers were observed for multiple sample groups, indicating differences in the ability and time course of vaccine induction of MenB (NadA) binding antibodies in different sequences, doses and formulations.

As can be seen from FIG. 18, the antibody-binding titers of each sample group were generally lower at 14 and 21 days after administration, which indicates that the immune response was weaker, the antibody titers of some sample groups began to rise significantly at 35 days after administration, and higher levels of antibody titers of multiple sample groups appeared at 49 and 70 days after administration, which indicates that the vaccine induction of different sequences, doses and formulations had differences in the ability and time course of production of MenB (NHBA) binding antibodies.

It can be seen from fig. 19 that the antibody-binding titers of each sample group were generally lower at 14 days and 21 days after administration, which means that the immune response was weaker at this time, that the antibody titers of a part of the sample groups began to rise significantly at 35 days after administration, and that the antibody titers of a plurality of sample groups appeared to reach higher levels at 49 days and 70 days after administration, which means that the vaccine induction of different sequences, doses and preparation forms had differences in the ability and time course of production of MenB (fHbp) binding antibodies.

4.3. Immune cell typing assay (FACs):

Spleen cells of mice with the MenB vaccine for prevention provided in injection examples 7, 8, 9,10, 11 and 12 were examined for T cell activation, and after stimulation with polypeptide, the spleen cells were stained with IFN-gamma/CD 69 dye and analyzed for T cell activation by FACs method, and test results of 34 days and 70 days after injection were recorded as shown in FIGS. 12-13.

1. Mu.g, 5. Mu.g and 15. Mu.g of the self-replicating mRNA sequence SEQ ID NO:15 provided in example 1 were injected into mice, 1. Mu.g, 5. Mu.g and 15. Mu.g of the self-replicating mRNA sequence SEQ ID NO:17 provided in example 2 were injected into mice, 1. Mu.g, 5. Mu.g and 15. Mu.g of the SEQ ID NO: 15-split-stock solution mixture provided in example 13 were injected into mice, 1. Mu.g, 5. Mu.g and 15. Mu.g of the split-preparation mixture provided in example 14 were injected into mice, 1. Mu.g, 5. Mu.g and 15. Mu.g of the SEQ ID NO: 17-split-stock solution mixture provided in example 15 were injected into mice, and the split-preparation mixture provided in example 16 was 1. Mu.g, 5. Mu.g and 15. Mu.g were injected into mice, and the results of the immunocyte typing assays were recorded after the injections, and the test results are shown in FIGS. 20-23.

FIG. 20 shows the result of CD4+IFNg+T cell typing detection, FIG. 21 shows the result of CD4+CD69+T cell typing detection, FIG. 22CD8+IFNg+T cell typing detection, and FIG. 23 shows the result of CD8+CD69+T cell typing detection.

It can be seen from FIGS. 20-23 that the vaccine of different samples can induce activation of CD4+ and CD8+ T cell subsets and change of related indexes of functions to a certain extent after administration, and the frequencies of CD4+IFNgamma+ T, CD4+CD69+ T, CD8 +IFNgamma+ T, CD8+CD69+ T cells of different sample groups are all increased, which indicates that the vaccine can activate T cell immune response of an organism, promote T cells to secrete cytokines or enter an early activation state and participate in immune defense. The vaccine samples of SEQ ID NO.15 sequence and SEQ ID NO.17 sequence with different dosages, and split-stock mixture and split-preparation mixture with different dosages have different capabilities of inducing T cell immune response and time courses, and the T cell immune response index induced by most sample groups shows an enhancement trend from 35 days to 70 days along with the increase of days after administration, so that the cell immune response of an organism to the vaccine is a dynamic process and is gradually enhanced within a period of time after administration.

IFN-gamma cytokine secretion level assay (ELISOPT):

Spleen cells from mice injected with the preventive MenB vaccine provided in examples 7, 8, 9, 10, 11 and 12 were used to detect IFN- γ secretion using the ELISPOT method. Preparing a mouse cell suspension from spleen cells, adding polypeptide for stimulation to enable the spleen cells to secrete IFN-gamma, adding avidin-enzyme complex, combining with biotin, adding chromogenic substrate to form visible spots, and recording ELISOPT test results of 34 days and 70 days of injection, wherein the test results are shown in figures 14-15.

1. Mu.g, 5. Mu.g and 15. Mu.g of the self-replicating mRNA sequence SEQ ID NO:15 provided in example 1 were injected into mice, 1. Mu.g, 5. Mu.g and 15. Mu.g of the self-replicating mRNA sequence SEQ ID NO:17 provided in example 2 were injected into mice, 1. Mu.g, 5. Mu.g and 15. Mu.g of the SEQ ID NO: 15-split-stock solution mixture provided in example 13 were injected into mice, and 1. Mu.g, 5. Mu.g and 15. Mu.g of the SEQ ID NO:15 split-preparation mixture provided in example 14 were injected into mice, and ELISOPT test results after the injection were recorded as shown in FIGS. 24 to 25.

FIG. 24 shows the results of ELISPOT assay at day D35 and FIG. 25 shows the results of ELISPOT assay at day D70.

The results of detecting protein bands by immunoblotting (WesternBlotting) provided in FIG. 7 show that 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 appear a target band at the corresponding positions, indicating that the 6 sequences can normally express the target protein in the cell. The results of binding antibodies in FIGS. 8-11 show that the antibody titers of SEQ ID NO:15 and SEQ ID NO:17 were highest. The immune cell types of the groups in FACs detection results after injection of the preventive MenB vaccine are shown in FIGS. 12-13 without significant difference, and the ELISPOT results after injection of the preventive MenB vaccine are shown in FIGS. 14-15 to be slightly higher in the SEQ ID NO:17 group and the SEQ ID NO:19 group. From the results presented by the intracellular protein expression, in vivo binding antibody data, FACs detection data and ELISPOT results, the self-replicating mRNA sequence designed by the application can induce immune response against meningococcus in vivo, wherein the application experiment effect of SEQ ID NO:17 group is the best.

As can be seen from fig. 24 and 25, on D35 and D70 days, different sample groups all detected a certain amount of cell secretion cytokines (as represented by SFU values) under the stimulation of multiple antigens (fHbp, NHBA, nadA, porA), which indicates that vaccine samples with different sequences, doses and preparation forms can activate immune cells of the organism to a certain extent, promote secretion of cytokines and initiate immune responses.

4.5 Serum bactericidal titer detection:

The resuscitated strain is incubated in a 5% CO ₂ environment at 37℃for 16-24h. About 20-40 colonies were subcultured, incubated in a 37 ℃ 5% CO ₂ environment for 4 hours, bacterial suspensions were prepared, and adjusted to the appropriate concentrations. Serum to be tested is inactivated and diluted in a 96-well U-bottom microtiter plate with a sterile buffer. Sequentially adding the mixture of the preparation of the separation of SEQ ID NO. 15 and the mixture of the preparation of the separation of SEQ ID NO. 17 into diluted serum respectively as complement and bacterial suspension, and adding the corresponding mixture of the preparation of the separation of SEQ ID NO. 15 or the mixture of the preparation of the separation of SEQ ID NO. 17 and bacterial suspension into complement control holes and serum control holes. The plates were blocked and incubated at 37 ℃ for 60 minutes. mu.L of each well was removed and inoculated onto Columbia blood agar plates using an inclined method. Plates were incubated overnight at 37 ℃ in 5% CO ₂ environment and colonies were counted. The bactericidal titer of the sample to be tested is the highest dilution multiple with the viable complement well as a reference and the colony count is less than 50% of the viable complement well, the test results of the SEQIDNO 17 resolution-preparation mixture are shown in FIG. 26, and the test results of the SEQIDNO 15 resolution-preparation mixture are shown in FIG. 27.

From FIG. 26, it can be seen that the serum bactericidal titer corresponding to the split-preparation mixture of SEQ ID NO:17 is 1:32, meaning that a certain amount of bacteria can still be effectively killed when the serum is diluted to 1:32, the bactericidal capacity is reduced and the bacterial colonies on the flat plate are obviously increased above the dilution.

From FIG. 27, it can be seen that the serum bactericidal titer corresponding to the split-preparation mixture of SEQ ID NO:15 is 1:256, meaning that a certain amount of bacteria can still be effectively killed when the serum is diluted to 1:256, and the bactericidal capacity is reduced and the bacterial colonies on the flat plate are increased above the dilution.

Claims

1. The self-replicating mRNA sequence is characterized by comprising an mRNA sequence encoding an amino acid sequence, wherein 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;

The 5' end of the tandem sequence is added with 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;

The single sequences in the series sequences are connected by using a flexible Linker in an amino acid sequence SEQ ID NO. 11;

the end of the tandem sequence is added with MITD molecular sequences, and the MITD molecular sequences comprise an amino acid sequence SEQ ID NO. 13;

the self-replicating mRNA sequence includes target protein amino acids designed in tandem for fHbp, NHBA, nadA and PorA antigen proteins.

2. The self-replicating mRNA sequence according to claim 1, wherein the sequence of the self-replicating mRNA comprises an mRNA sequence encoding the corresponding amino acid sequence SEQ ID No. 17.

3. A prophylactic MenB vaccine comprising a self-replicating mRNA sequence according to any one of claims 1-2.

4. A method of preparing a prophylactic MenB vaccine according to claim 3, comprising the steps of: