Pertussis mRNA vaccine

Abstract

The invention provides a pertussis mRNA vaccine, and belongs to the technical field of biology. Antigens of the pertussis mRNA vaccine comprise pertussis toxin and filamentous hemagglutinin, a gene sequence of the pertussis toxin comprises gS1 and gC180, ninth arginine of amino acid sequences corresponding to the gS1 and the gC180 mutates into lysine, and 129th glutamic acid of the amino acid sequences corresponding to the gS1 and the gC180 mutates into glycine. A signal peptide of a human Igk light chain is added to the upstream of the amino acid sequence to replace a natural bacterial signal peptide; the gene sequence of the filamentous hemagglutinin comprises an FHA456 gene, an FHA373 gene and an FHA233 gene. According to the invention, the sequences for coding the antigen genes gS1, gC180, FHA456, FHA373 and FHA233 are optimized, so that an organism can be efficiently induced to generate immune response.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a pertussis mRNA vaccine. Background Art

[0002] Pertussis is an acute respiratory infectious disease caused by Bordetella pertussis. Because its early symptoms are not typical, it is not easy to diagnose, and it is highly contagious, so it is difficult to control the spread and often causes epidemics. It was once one of the main causes of morbidity and mortality in infants and young children. Currently used pertussis vaccines include whole cell pertussis vaccine (wP) and acellular pertussis vaccine (aP). After vaccination with wP vaccine, the incidence and mortality of pertussis have been greatly reduced, but adverse reactions are likely to occur after vaccination. The main components of aP vaccine are highly purified filamentous haemagglutinin (FHA), chemically detoxified pertussis toxin (PT), and pertactin (PRN), etc. The adverse reactions are significantly lower than those of wP vaccine, and the willingness and compliance of the vaccinated are high, and the vaccination coverage rate of the vaccine is relatively high. However, despite the high vaccination coverage rate of aP vaccine, the incidence of pertussis has shown a significant upward trend in the past 20 years, showing the phenomenon of "pertussis recurrence". One of the reasons for this phenomenon may be that the protective effect of aP vaccine is weaker than that of wP vaccine. Research shows that although aP vaccine induces a certain degree of cellular immune response, the type of immune response shifts towards antibody immunity mediated by Th2 cells; while the type of immune response induced by natural infection and wP vaccine is mainly Th1 cells and Th17 cells. The immune response produced by aP vaccine tends to neutralize toxins and prevent bacterial adhesion, and the inability to induce cellular immune responses such as phagocytosis and killing of intracellular bacteria similar to those after natural infection is one of the important reasons for pertussis recurrence. mRNA vaccines are flexible in design, fast in R & D speed, do not involve bacterial culture in the vaccine development process, and have high safety. mRNA vaccines can simultaneously induce high levels of humoral and cellular immune responses, and can be used to develop new pertussis vaccines, which is an effective strategy to deal with pertussis recurrence. However, current mRNA vaccines have technical problems such as unstable chemical properties, easy degradation, and insufficient protein expression. Summary of the Invention

[0003] In view of this, the present invention provides a pertussis mRNA vaccine, which optimizes the sequences of genes encoding gS1, gC180, FHA456, FHA373, and FHA233 antigens, and can efficiently induce the body to produce an immune response.

[0004] To achieve the above object, the present invention adopts the following technical solutions: The present invention provides a pertussis mRNA vaccine. The antigens of the pertussis mRNA vaccine include pertussis toxin and filamentous hemagglutinin. The gene sequence of the pertussis toxin includes gS1 and gC180. The gS1 is the gene sequence of the S1 subunit of the pertussis toxin, and the gC180 is the gene sequence corresponding to 180 amino acids at the N-terminus of the S1 subunit of the pertussis toxin. The arginine at the 9th position and the glutamate at the 129th position of the amino acid sequences corresponding to the gS1 and the gC180 are mutated into lysine and glycine respectively, and a signal peptide of the human Igk light chain is added upstream of their amino acid sequences to replace the natural bacterial signal peptide; for the filamentous hemagglutinin, its gene sequence includes FHA456, FHA373, and FHA233, and the FHA456, FHA373, and FHA233 respectively correspond to positions 1655 to 2111, 1545 to 1917, and 1655 to 1917 of the amino acid sequence of the filamentous hemagglutinin.

[0005] Preferably, the mRNA encoding the gS1 antigen gene contains a nucleotide sequence having at least 85%, at least 90%, at least 95%, or 100% identity with the nucleotide sequences shown in SEQ ID NO: 1-SEQ ID NO: 6.

[0006] Preferably, the mRNA encoding the gC180 antigen gene contains a nucleotide sequence having at least 85%, at least 90%, at least 95%, or 100% identity with the nucleotide sequences shown in SEQ ID NO: 7-SEQ ID NO: 12.

[0007] Preferably, the mRNA encoding the FHA456 antigen gene contains a nucleotide sequence having at least 85%, at least 90%, at least 95%, or 100% identity with the nucleotide sequences shown in SEQ ID NO: 13-SEQ ID NO: 18.

[0008] Preferably, the mRNA encoding the FHA373 antigen gene contains a nucleotide sequence having at least 85%, at least 90%, at least 95%, or 100% identity with the nucleotide sequences shown in SEQ ID NO: 19-SEQ ID NO: 24.

[0009] Preferably, the mRNA encoding the FHA233 antigen gene contains a nucleotide sequence having at least 85%, at least 90%, at least 95%, or 100% identity with the nucleotide sequences shown in SEQ ID NO: 25-SEQ ID NO: 30.

[0010] Preferably, the gene sequence of the DNA molecule encoding the gS1 antigen gene is as shown in SEQ ID NO: 31-SEQ ID NO: 36; and / or, A DNA molecule encoding the gC180 antigen gene, the sequence of which is shown in SEQ ID NO: 37 - SEQ ID NO: 42; and / or, A DNA molecule encoding the FHA456 antigen gene, the sequence of which is shown in SEQ ID NO: 43 - SEQ ID NO: 48; and / or, A DNA molecule encoding the FHA373 antigen gene, the sequence of which is shown in SEQ ID NO: 49 - SEQ ID NO: 54; and / or, A DNA molecule encoding the FHA233 antigen gene, the sequence of which is shown in SEQ ID NO: 55 - SEQ ID NO: 60.

[0011] Preferably, the mRNA molecules encoding the gS1, gC180, FHA456, FHA373, and FHA233 antigen genes all include: (a) A 5'-cap structure, preferably a Cap1 structure; (b) A 5' untranslated region (5'-UTR); (c) A Kozak sequence; (d) A Bordetella pertussis antigen gene sequence encoding pertussis toxin and filamentous hemagglutinin antigens capable of inducing an immune response; (e) A 3' untranslated region (3'-UTR); (f) A polyadenylate tail element with a total length of 100 nt or more.

[0012] Preferably, an antigen carrier is included, and the antigen carrier is used to encapsulate the antigen of the Bordetella pertussis mRNA vaccine.

[0013] Preferably, the antigen carrier includes ionizable lipids, neutral lipids, PEGylated lipids, and cholesterol.

[0014] Compared with the prior art, the beneficial effects of the present invention are: (1)The present invention optimizes the sequences of the antigen genes encoding gS1, gC180, FHA456, FHA373, and FHA233. The sequence optimization includes the structural stability of mRNA, the usage frequency of common human codons, various cis-elements, and codon adaptability, etc. Their DNA sequences are shown in SEQ ID NO:31-SEQ ID NO:36, SEQ ID NO:37-SEQ ID NO:42, SEQ ID NO:43-SEQ ID NO:48, SEQ ID NO:49-SEQ ID NO:54, and SEQ ID NO:55-SEQ ID NO:60 respectively. Using the optimized DNA as a template (linearized plasmid) for transcription, mRNA molecules encoding Bordetella pertussis gS1, gC180, FHA456, FHA373, and FHA233 antigen genes are obtained, and their sequences are shown in SEQ ID NO:1-SEQ ID NO:6, SEQ ID NO:7-SEQ ID NO:12, SEQ ID NO:13-SEQ ID NO:18, SEQ ID NO:19-SEQ ID NO:24, and SEQ ID NO:25-SEQ ID NO:30 respectively. The optimized nucleotide sequences make the transcribed mRNA structure more stable, with higher translation efficiency of the target protein in mammals and humans, and can solve the technical problems such as the unstable chemical properties of mRNA, easy degradation, and insufficient protein expression in the prior art, and can efficiently induce the body to produce an immune response.

[0015] (2)The present invention replaces all uracils (U) in the mRNA nucleic acid molecule with pseudouridine (ψ), reducing the content of uracil in the mRNA molecule and decreasing the recognition of mRNA by the immune system.

[0016] (3)The present invention encapsulates the antigens of the Bordetella pertussis mRNA vaccine with an antigen carrier to protect the nucleic acid from degradation and facilitate cell uptake and mRNA release. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is an expression verification diagram of in vitro transcribed gC180-mRNA, gS1-mRNA, and S1-mRNA transfected into HEK293T cells provided by Example 2 of the present invention; Figure 2 It is an expression verification diagram of in vitro transcribed FHA456-mRNA, FHA373-mRNA, and FHA233-mRNA transfected into HEK293T cells provided by Example 2 of the present invention; Figure 3Cytotoxicity verification diagrams of in vitro transcribed gC180-mRNA, gS1-mRNA, and S1-mRNA transfected into CHO cells provided in Example 3 of the present invention; Figure 4 Diagrams of the survival rates of NIH mice observed after immunization with gC180-mRNA-LNP and gS1-mRNA-LNP provided in Example 5 of the present invention for 14 days; Figure 5 Diagrams of the survival rates of NIH mice observed after immunization with gC180-mRNA-LNP+FHA456-mRNA-LNP, gC180-mRNA-LNP+FHA373-mRNA-LNP, and gC180-mRNA-LNP+FHA233-mRNA-LNP provided in Example 5 of the present invention for 14 days; Figure 6 Diagrams of the PT antibody levels in the sera of CD1 mice detected by ELISA after immunization with gC180-mRNA-LNP vaccine and gS1-mRNA-LNP provided in Example 6 of the present invention; Figure 7 Diagrams of the PT antibody levels in the sera of CD1 mice detected by ELISA after immunization with gC180-mRNA-LNP+FHA456-mRNA-LNP, gC180-mRNA-LNP+FHA373-mRNA-LNP, and gC180-mRNA-LNP+FHA233-mRNA-LNP provided in Example 6 of the present invention; Figure 8 Diagrams of the FHA antibody levels in the sera of CD1 mice detected by ELISA after immunization with gC180-mRNA-LNP+FHA456-mRNA-LNP, gC180-mRNA-LNP+FHA373-mRNA-LNP, and gC180-mRNA-LNP+FHA233-mRNA-LNP provided in Example 6 of the present invention. Detailed implementation manners

[0018] The present invention will be further described in detail below in conjunction with specific embodiments, so that those skilled in the art can understand the present invention more clearly.

[0019] The present invention provides a method for constructing a transcription template based on homologous recombination technology, and the specific operation steps are as follows: Preparation of the basic plasmid template: First, select the basic plasmid template containing SEQ ID NO:31 - SEQ ID NO:36, SEQ ID NO:37 - SEQ ID NO:42, SEQ ID NO:43 - SEQ ID NO:48, SEQ ID NO:49 - SEQ ID NO:54, SEQ ID NO:55 - SEQ ID NO:60. This plasmid template can provide a vector backbone (target gene) suitable for PCR amplification and contains necessary functional sequences to support subsequent homologous recombination and transcription reactions.

[0020] PCR amplification of the target gene: Use PCR technology to amplify the target gene from the above basic plasmid template. The primers used should be designed to ensure that homologous sequences corresponding to the respective regions on the cloning vector can be obtained at both ends of the target gene, thus supporting subsequent homologous recombination. The PCR product needs to be confirmed for the size and purity of the amplified fragment by electrophoresis.

[0021] Homologous recombination operation: Co - perform homologous recombination on the target gene fragment obtained by PCR amplification and the cloning vector containing the UTR (untranslated region) sequence and a 120 - nucleotide (nt) polyadenylate tail. In this process, the target gene is precisely ligated into the plasmid template through homologous recombination technology to construct a transcription template with an ideal sequence structure. It should be noted that the UTR sequence and the polyadenylate tail can effectively promote gene expression and RNA stability after transcription.

[0022] Transformation and sequence verification: The recombinant plasmid after homologous recombination is transformed into TOP10 competent Escherichia coli cells and cultured using conventional transformation methods. After transformation, single clones containing the recombinant plasmid are screened, and the plasmid sequence constructed is verified by PCR and sequencing methods to check if it is consistent with the expected sequence. Confirmation of the sequencing results is a crucial step to ensure successful construction and exclude possible incorrect sequences.

[0023] Fermentation of the strain and plasmid extraction: After the sequence is confirmed to be correct, the Escherichia coli strain containing the target recombinant plasmid is cultured by shake - flask fermentation. By optimizing the culture conditions, ensure that the strain can grow efficiently and produce a sufficient amount of recombinant plasmid. During fermentation, control factors such as temperature, pH value, and shake - flask rotation speed to maximize plasmid production.

[0024] Plasmid purification: Use an endotoxin - free large - scale plasmid extraction kit to extract the fermentation broth and purify high - quality recombinant plasmid. The plasmid extraction process is strictly operated according to the kit instructions to ensure that the obtained plasmid has high purity and no endotoxin contamination. The finally extracted plasmid can be used as a transcription template for subsequent in vitro transcription experiments, further for functional research or production applications. The following content is the specific method.

[0025] Example 1 Linearization, in vitro transcription and capping of plasmid 1.1 Linearization of plasmid, the system is shown in the following table:

[0026] Incubate at 37 °C for 4 hours.

[0027] 1.2 Purification of linearized plasmid Use Tiangen Biochemical Universal DNA Purification and Recovery Kit (DP214) to purify the enzyme digestion product. The process is as follows: First, place the adsorption column in the collection tube. Add 100 μL of PC to the enzyme digestion product, pipette and mix well, then transfer it to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid. Add 600 μL of washing solution PW, centrifuge at 12000 rpm for 1 min, and discard the waste liquid again. Repeat once. Place the adsorption column in a clean 1.5 mL centrifuge tube, add 30 μL of ddH2O, let it stand for 1 min, centrifuge at 12000 rpm for 1 min, and store the collected solution at -20 °C for later use. Use NanoDrop to measure the concentration of the linearized plasmid.

[0028] 1.3 In vitro transcription Use the commercial mRNA IVT kit, High-yield T7 in vitro transcription reagent (N1-Me-pUTP) Hi-yield T7 in vitro transcription reagent (N1-Me-pUTP)-HBP001505 to prepare mRNA. Operate according to the kit instructions with the obtained linearized plasmid. The system is as follows: The reaction system is:

[0029] React at 37 °C for 4 h, then add DNase I to the reaction system and react at 37 °C for 15 min to digest the transcribed DNA template.

[0030] 1.4 Lithium chloride purification Lithium chloride purification can remove proteins and most free nucleotides. Add 20 μL of lithium chloride precipitation solution (5 M lithium chloride) to the previous step of the transcription product. Gently pipette and mix well, then incubate at -20 °C for 30 min, centrifuge at 12000 rpm for 15 min, and discard the supernatant. Add 500 μL of pre-cooled 70% ethanol to wash the RNA precipitate, centrifuge at 4 °C and 12000 rpm for 15 min, and discard the supernatant. Repeat twice. Open the lid and dry for 2 min, add 20 - 50 μL of RNase-free ddH2O to dissolve the RNA precipitate.

[0031] 1.5 Capping First, heat the purified uncapped mRNA at 65°C for 10 min, and then quickly ice-bath for 5 min.

[0032] Then, use the enzyme capping reagent Cap1 Capping System-HBP001513 from Hanhai New Enzyme for capping. The reaction system is as follows:

[0033] Reaction conditions: React at 37°C for 60 min.

[0034] 1.6 Then use the method in 1.4 for lithium chloride purification.

[0035] Example 2 Verification of mRNA transfection and expression in HEK293T cells Twenty-four hours before transfection, seed HEK293T cells into a 6-well plate at a density of 4×10 5 cells / well. The culture medium is DMEM:VP = 1:2 complete medium (5% FBS). Transfect HEK293T cells using the Lipomaster 2000 Transfection Reagent, with 2 μg of mRNA transfected per well. After culturing in an incubator at 37°C for 24 hours, detect the protein products in the cell lysates and cell supernatants. For the cell fraction, gently wash the cells with phosphate-buffered saline (PBS) first, then add cell lysis buffer to lyse the cells, and centrifuge to collect the supernatant for use. For the cell supernatant, ultrafilter it using a 10 kd ultrafiltration centrifugal tube, add an appropriate amount of PBS and ultrafilter several times, and place the ultrafiltered cell supernatant in an EP tube for use. Add an appropriate amount of the above samples to a protein sample buffer containing β-mercaptoethanol and boil for 5 minutes. Perform 10-well sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a concentration of 4-12%, load 20 μL per well, and the electrophoresis conditions are 80V for 30 min first, and then 120V for 120 min. After electrophoresis, transfer the proteins to an NC membrane using an automatic rapid wet transfer instrument, and block it with PBS containing 5% skim milk powder at room temperature for 1 h. Add PBST and wash for 5 min, add antibody 1B7-HRP or FHA polyclonal antibody-HRP (1:5000), shake for 30 min, and incubate at 37°C for 1 h. After incubation, wash 5 times with PBST, add Immobilon Western HRP substrate chromogenic solution and use a gel imager for exposure and photography.

[0036] Results: As Figure 1 shown, after mRNA transfection of HEK293T cells, gC180, gS1, and S1 proteins are produced and specifically bind to the monoclonal antibody 1B7 against the PT-S1 subunit, while no corresponding target protein bands are produced in the untransfected cell samples. As Figure 2As shown, after mRNA transfection of HEK293T cells, FHA456-mRNA, FHA373-mRNA, and FHA233-mRNA proteins were produced and specifically bound to the polyclonal antibody against FHA, while no corresponding target protein bands were produced in the untransfected cell samples. The above results indicate that the constructed gC180-mRNA, gS1-mRNA, S1-mRNA, FHA456-mRNA, FHA-373mRNA, and FHA233-mRNA can efficiently transfect cells and effectively express the target proteins.

[0037] Example 3 Cytotoxicity Detection of CHO Cells Active PT has the effect of clustering Chinese Hamster Ovary (CHO) cells. gC180-mRNA, gS1-mRNA, and S1-mRNA were transfected into CHO cells in the same way, and the clustering status of CHO cells was monitored within 72 hours.

[0038] As Figure 3 shown, no clustering phenomenon occurred in CHO cells transfected with gene-detoxified gC180-mRNA and gS1-mRNA, while obvious clustering phenomenon occurred in CHO cells transfected with non-gene-detoxified C180-mRNA.

[0039] Example 4 Preparation of mRNA-LNP Ionizable lipid, neutral lipid, PEGylated lipid, and cholesterol were dissolved in absolute ethanol at a ratio of 50:2.5:10:37.5, and the prepared gC180-mRNA was diluted with 50 mmol / mL citrate buffer (pH 4.0). The lipid mixture and the diluted mRNA were prepared into mRNA-LNP at a ratio of 1:3 at a speed of 12 mL / min through a nano-drug preparation system; the prepared product was diluted 25-fold with DPBS, and the alcohol was removed by changing the solution through a 30KDa ultrafiltration centrifugal tube, and then sterilized by filtration through a 0.22μm filter membrane to obtain mRNA-LNP, and the particle size was analyzed by a particle size analyzer. The mRNA-LNP was broken by 1% Triton100 at 37°C for 10 min, and the concentration of RNA before and after breaking was measured using the Qubit RNA XR Aassy kit, and the encapsulation efficiency of mRNA was calculated.

[0040] Encapsulated mRNA concentration = Total mRNA concentration in the broken sample - mRNA concentration in the unbroken sample Encapsulation efficiency = (Encapsulated mRNA concentration / Total mRNA concentration in the broken sample) × 100% Results: The prepared mRNA-LNP was a transparent solution with a faint blue opalescence. The average particle size of the mRNA-LNP was about 80 nm, the PDI was 0.05, and the sample had good homogeneity. The encapsulation efficiency of the sample detected by the Qubit fluorescence dye method was 97.5%.

[0041] Example 5 Immunoprotective Evaluation NIH mice, weighing 10 - 12 g and with an equal number of males and females, 20 mice in each group, were used. At the same time, empty-encapsulated LNP was set as a negative control. NIH mice were immunized intraperitoneally with 500 μL per mouse of 10 μg of gC180-mRNA-LNP and gS1-mRNA-LNP respectively. 21 days after immunization, each mouse was challenged intracranially with 0.03 mL of Bordetella pertussis CMCC 58030 (18323) bacterial solution (containing 8.0×10 4 ) using a 0.25 mL syringe. The number of dead mice within 14 days was observed and recorded.

[0042] NIH mice, weighing 10 - 12 g and with an equal number of males and females, 20 mice in each group, were used. At the same time, empty-encapsulated LNP was set as a negative control. NIH mice were immunized intraperitoneally with 500 μL per mouse of 5 μg of gC180-mRNA-LNP combined with 5 μg of FHA456-mRNA-LNP, 5 μg of gC180-mRNA-LNP combined with 5 μg of FHA373-mRNA-LNP, and 5 μg of gC180-mRNA-LNP combined with 5 μg of FHA233-mRNA-LNP respectively. 21 days after immunization, each mouse was challenged intracranially with 0.03 mL of Bordetella pertussis CMCC 58030 (18323) bacterial solution (containing 8.0×10 4 ) using a 0.25 mL syringe. The number of dead mice within 14 days was observed and recorded.

[0043] Results: As Figure 4 shown, the survival rate of mice immunized with 10 μg of gC180-mRNA-LNP was 31.25%, the survival rate of mice immunized with 10 μg of gS1-mRNA-LNP was 12.50%, the survival rate of mice immunized with chemically detoxified dPT + Al(OH)3 adjuvant was 62.5%, while all mice in the empty-encapsulated LNP immunization group died.

[0044] As Figure 5As shown, the survival rate of mice immunized with 5 μg gC180-mRNA-LNP and 5 μg FHA456-mRNA-LNP was 56.25%, the survival rate of mice immunized with 5 μg gC180-mRNA-LNP and 5 μg FHA373-mRNA-LNP was 37.5%, the survival rate of mice immunized with 5 μg gC180-mRNA-LNP and 5 μg FHA233-mRNA-LNP was 50%, the survival rate of mice immunized with chemically detoxified dPT+FHA+Al(OH)3 adjuvant was 81.25%, while all the mice in the empty LNP immunization group died.

[0045] Example 6 Immunogenicity Detection Female CD1 mice weighing 20 - 24 g, 10 mice in each group, were intraperitoneally immunized with 10 μg gC180-mRNA-LNP and gS1-mRNA-LNP respectively, 500 μL per mouse. Meanwhile, empty LNP was set as a negative control. Four weeks after immunization, whole blood was collected by eye bleeding. The PT antibody level was detected by ELISA, and the international standard product of ELISA was NIBSC 97 / 642.

[0046] Female CD1 mice weighing 20 - 24 g, 10 mice in each group, were intraperitoneally immunized with 5 μg gC180-mRNA-LNP and 5 μg FHA456-mRNA-LNP, 5 μg gC180-mRNA-LNP and 5 μg FHA373-mRNA-LNP, and 5 μg gC180-mRNA-LNP and 5 μg FHA233-mRNA-LNP respectively, 500 μL per mouse. Meanwhile, empty LNP was set as a negative control. Four weeks after immunization, whole blood was collected by eye bleeding. The PT antibody level and FHA antibody level were detected by ELISA, and the international standard product of ELISA was NIBSC 97 / 642.

[0047] Results: As Figure 6 shown, there were significant differences in the production of anti-PT IgG antibodies in mice of the gC180-mRNA-LNP and gS1-mRNA-LNP groups.

[0048] As Figure 7 、 8As shown, compared with the LNP of the empty package, there were significant differences in the production of anti-PT IgG antibodies and anti-FHA IgG antibodies in the mice of the gC180-mRNA-LNP+FHA456-mRNA-LNP, gC180-mRNA-LNP+FHA373-mRNA-LNP, and gC180-mRNA-LNP+FHA233-mRNA-LNP groups; there were no significant differences in the production of anti-PT IgG antibodies among the mice in the gC180-mRNA-LNP+FHA456-mRNA-LNP, gC180-mRNA-LNP+FHA373-mRNA-LNP, and gC180-mRNA-LNP+FHA233-mRNA-LNP groups, but there were significant differences in the production of anti-FHA IgG antibodies.

[0049] In the present invention, the specific raw materials not described are all existing substances and can be directly purchased from the market.

[0050] The above are only the preferred implementation schemes of the present invention and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A pertussis mRNA vaccine, characterized in that, The antigens of the pertussis mRNA vaccine include: Pertussis toxin, whose gene sequence includes gS1 and gC180. The gS1 is the gene sequence of the S1 subunit of pertussis toxin, and the gC180 is the gene sequence corresponding to 180 amino acids at the N-terminus of the S1 subunit of pertussis toxin. The arginine at the 9th position and the glutamate at the 129th position in the amino acid sequences corresponding to the gS1 and the gC180 are mutated into lysine and glycine respectively, and a signal peptide of human Igk light chain is added upstream of their amino acid sequences to replace the natural bacterial signal peptide; and, Filamentous hemagglutinin, whose gene sequence includes FHA456, FHA373, and FHA233. The FHA456, FHA373, and FHA233 respectively correspond to positions 1655 to 2111, 1545 to 1917, and 1655 to 1917 of the amino acid sequence of filamentous hemagglutinin.

2. The pertussis mRNA vaccine according to claim 1, wherein The mRNA encoding the gS1 antigen gene contains a nucleotide sequence having at least 85%, at least 90%, at least 95%, or 100% identity with the nucleotide sequences shown in SEQ ID NO:1 - SEQ ID NO:

6.

3. The pertussis mRNA vaccine according to claim 2, characterized in that, The mRNA encoding the gC180 antigen gene contains a nucleotide sequence having at least 85%, at least 90%, at least 95%, or 100% identity with the nucleotide sequences shown in SEQ ID NO:7 - SEQ ID NO:

12.

4. The pertussis mRNA vaccine according to claim 3, characterized in that, The mRNA encoding the FHA456 antigen gene contains a nucleotide sequence having at least 85%, at least 90%, at least 95%, or 100% identity with the nucleotide sequences shown in SEQ ID NO:13 - SEQ ID NO:

18.

5. The pertussis mRNA vaccine according to claim 4, wherein, The mRNA encoding the FHA373 antigen gene contains a nucleotide sequence having at least 85%, at least 90%, at least 95%, or 100% identity with the nucleotide sequences shown in SEQ ID NO:19 - SEQ ID NO:

24.

6. The pertussis mRNA vaccine according to claim 5, wherein The mRNA encoding the FHA233 antigen gene contains a nucleotide sequence having at least 85%, at least 90%, at least 95%, or 100% identity with the nucleotide sequences shown in SEQ ID NO:25 - SEQ ID NO:

30.

7. The pertussis mRNA vaccine according to claim 1, wherein The gene sequence of the DNA molecule encoding the gS1 antigen gene is as shown in SEQ ID NO:31 - SEQ ID NO:36; and / or, The DNA molecule encoding the gC180 antigen gene, whose sequence is as shown in SEQ ID NO:37 - SEQ ID NO:42; and / or, The DNA molecule encoding the FHA456 antigen gene, whose sequence is as shown in SEQ ID NO:43 - SEQ ID NO:48; and / or, The DNA molecule encoding the FHA373 antigen gene, whose sequence is as shown in SEQ ID NO:49 - SEQ ID NO:54; and / or, The DNA molecule encoding the FHA233 antigen gene, whose sequence is as shown in SEQ ID NO:55 - SEQ ID NO:

60.

8. The pertussis mRNA vaccine according to claim 7, characterized in that, The mRNA molecules encoding the gS1, gC180, FHA456, FHA373, and FHA233 antigen genes all include: (a) a 5'-cap structure; (b) a 5'-untranslated region; (c) a Kozak sequence; (d) a Bordetella pertussis antigen gene sequence encoding pertussis toxin and filamentous hemagglutinin antigens capable of inducing an immune response; (e) a 3'-untranslated region; (f) a polyadenylate tail element with a total length of 100 nt or more.

9. The pertussis mRNA vaccine according to claim 1, wherein An antigen carrier is included, and the antigen carrier is used to encapsulate the antigen of the Bordetella pertussis mRNA vaccine.

10. The pertussis mRNA vaccine according to claim 9, characterized in that, The antigen carrier includes ionizable lipids, neutral lipids, PEGylated lipids, and cholesterol.

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