Neisseria meningitidis recombinant protein and application thereof

Abstract

The invention discloses neisseria meningitidis recombinant protein and application thereof. In particular to a neisseria meningitidis recombinant protein, a preparation method thereof and application of the neisseria meningitidis recombinant protein in a vaccine composition, and the recombinant protein can induce a more efficient specific bactericidal antibody and can be used for preventing infection caused by a group B meningococcal strain.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering vaccine technology, and in particular to a recombinant protein of Neisseria meningitidis, a vaccine composition containing the recombinant protein, and its use in the preparation of a vaccine to prevent Neisseria meningitidis group B infection. Background Technology

[0002] Epidemic cerebrospinal meningitis (meningococcal meningitis) is a serious respiratory infectious disease caused by Neisseria meningitidis. Neisseria meningitidis is transmitted through respiratory or pharyngeal droplets from infected individuals; close or prolonged contact can lead to disease transmission. Statistics show that the global average mortality rate of meningococcal meningitis is approximately 10%. Even with timely treatment and the elimination of the pathogen from the body, about 15% of patients still experience neurological sequelae, severely impacting their quality of life.

[0003] Neisseria meningitidis is a Gram-negative bacterium. After Gram staining, it appears red under a microscope, and is diplococcal or spherical, occurring singly, in pairs, or in irregular arrangements of four. The bacteria are approximately 0.7 µm in diameter, and after culture, the colony morphology is spherical. Capsular polysaccharides (CPS) are located on the surface of Neisseria meningitidis and are composed of several monosaccharides linked to sialic acid or phosphate. When Neisseria meningitidis invades host cells, CPS helps resist the host's immune response and prevents the bacteria from adhering tightly to the host cells. Currently, based on the chemical structure and immunogenicity of CPS, Neisseria meningitidis can be classified into 13 serogroups: A, B, C, D, E, H, I, K, L, W135, X, Y, and Z. Among them, the prevalence of serogroup B Neisseria meningitidis (MenB) strains in my country is increasing year by year. However, the capsular polysaccharide (CPS) of serogroup B Neisseria meningitidis has weak immunogenicity and exhibits cross-reactivity; therefore, the development of a serogroup B meningococcal vaccine is a significant challenge in the current vaccine field.

[0004] In 2000, Pizza et al. screened 28 protein antigens that could effectively induce the production of bactericidal antibodies using reverse vaccinology methods. These included Neisserial adhesin A (NadA), Factor H-binding protein (FHbp), and Neisseria heparin binding antigen (NHBA), all of which could serve as candidate proteins for serogroup B meningococcal vaccines. FHbp can be divided into two subgroups, A and B. Subgroup A includes variants V2 and V3, while subgroup B includes variant V1. Five variants of NadA have been identified: NadA-1, NadA-2, NadA-3, NadA-4, and NadA-5.

[0005] Currently, there are two main serogroup B meningococcal vaccines on the international market. One is produced by GlaxoSmithKline (GSK) under the brand name Bexsero, whose main components are FHbp, NadA, NHBA, and OMV. The other is produced by Pfizer under the brand name Trumenba, which contains only two variants of the FHbp protein, V1.55 and V3.45. However, given the diversity and variability of the surface proteins of Neisseria meningitidis, selecting the appropriate protein has become a challenge in developing a serogroup B meningococcal vaccine. Summary of the Invention

[0006] The purpose of this invention is to provide a recombinant protein of Neisseria meningitidis, its preparation method, and its application.

[0007] In a first aspect, the present invention provides a recombinant protein of Neisseria meningitidis, the recombinant protein comprising Factor H Binding Protein Variant 1 (FHbp V1 variant), Factor H Binding Protein Variant 2 (FHbp V2 variant), and Factor H Binding Protein Variant 3 (FHbp V3 variant), wherein the amino acid sequence of Factor H Binding Protein Variant 1 is shown in SEQ ID NO: 1, the amino acid sequence of Factor H Binding Protein Variant 2 is shown in SEQ ID NO: 2, and the amino acid sequence of Factor H Binding Protein Variant 3 is shown in SEQ ID NO: 3.

[0008] In some embodiments, the recombinant protein further comprises GNA2091 or a heparin-binding antigen (NHBA protein).

[0009] In some embodiments, the amino acid sequence of GNA2091 is shown in SEQ ID NO: 4, and the amino acid sequence of the heparin-binding protein is shown in SEQ ID NO: 5 or SEQ ID NO: 6.

[0010] In some embodiments, the recombinant protein has an amino acid sequence as described in any of SEQ ID NO: 7 or SEQ ID NO: 9-23.

[0011] In a second aspect, the present invention provides another recombinant Neisseria meningitidis protein, characterized in that it is composed of a heparin-binding antigen (NHBA protein), wherein the amino acid sequence of the heparin-binding antigen is shown in SEQ ID NO:5 or SEQ ID NO:6.

[0012] In some embodiments, the recombinant protein has a sequence as shown in SEQ ID NO:24.

[0013] A third aspect of the present invention provides a nucleic acid molecule characterized in that it encodes a recombinant protein as described in any of the above descriptions.

[0014] In a fourth aspect, the present invention provides a recombinant expression vector, characterized in that it comprises any of the nucleic acid molecules described above.

[0015] In a fifth aspect, the present invention provides a host cell characterized in that it expresses a fusion protein as described above; or contains a nucleic acid molecule as described above; or contains a recombinant expression vector as described above.

[0016] A sixth aspect of the present invention provides a method for preparing recombinant proteins, characterized in that the method comprises:

[0017] The host cells described above are cultured to obtain a culture medium; and the recombinant protein is isolated from the culture medium.

[0018] In some embodiments, the host cell is selected from the group consisting of bacteria and fungi, preferably, the host cell is *Escherichia coli* Pichia pastoris; more preferably, the host cell is *Escherichia coli*.

[0019] A seventh aspect of the present invention provides a vaccine composition, characterized in that it comprises any of the recombinant proteins described above and a vaccinologically acceptable carrier, wherein the vaccinologically acceptable carrier comprises aluminum adjuvant, MF59, Freund's complete adjuvant, Freund's incomplete adjuvant, CpG adjuvant, BCG ribonucleic acid, or monophospholipase A.

[0020] Compared with the prior art, the present invention has the following beneficial effects: it can induce a more efficient specific bactericidal antibody response and can be used for the development of group B meningococcal vaccines. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of recombinant protein design. Among them, Figure 1 a is designed for FHbp class recombinant proteins. Figure 1 b is designed as a recombinant protein of FHbp-GNA2091. Figure 1 c represents the design of the FHbp-NHBA recombinant protein. Figure 1 d represents the design of NHBA recombinant protein. Detailed Implementation

[0022] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0024] the term

[0025] The term "and / or" includes any and all combinations of one or more of the related listed items.

[0026] In the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., the terms "first," "second," "third," "fourth," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly specifying the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only a non-exhaustive enumeration purpose and should be understood not to constitute a closed limitation on quantity.

[0027] The term "Factor H Binding Protein Variant 1 (FHbp V1 variant protein)" is a membrane surface lipoprotein. Many strains of meningococcus carry its gene, and strains that do not carry this gene have also been found (Lucidarme J, et al. Clin. Vaccine Immunol. 2011, 18(6):1002-1014.). Based on different amino acid sequences, this protein can be divided into three variants (V1 variant, V2 variant, and V3 variant). V2 variants and V3 variants have certain cross-protective effects, while the cross-protective effect between them and V1 variants is weak. Variant 1, Variant 2, and Variant 3 genes encode approximately 280 amino acid residues each. The amino acid homology of FHbp proteins of the same variant can be as low as about 90%, while the amino acid homology between FHbp proteins of different variants can be as low as 62%. The FHbp V1-V3 variant proteins used in this invention are not particularly limited and can be any wild-type or mutant FHbpV1-V3 variant protein. Preferably, the amino acid sequence of the FHbp V1 variant protein is as shown in SEQ ID NO:1, and may also include amino acid sequences in which one or more amino acids are added, deleted, or substituted from SEQ ID NO:1; the amino acid sequence of the FHbp V2 variant protein is as described in SEQ ID NO:2, and may also include amino acid sequences in which one or more amino acids are added, deleted, or substituted from SEQ ID NO:2; the amino acid sequence of the FHbp V3 variant protein is as described in SEQ ID NO:3, and may also include amino acid sequences in which one or more amino acids are added, deleted, or substituted from SEQ ID NO:3.

[0028] The term “GNA2091” is another antigen of group B Neisseria meningitidis, and its amino acid sequence is shown in SEQ ID NO: 4.

[0029] The term "Neisserial Heparin-Binding Antigen (NHBA protein)" refers to a lipoprotein specific to the genus Neisseria, expressed in almost all strains. However, the amino acid sequence of NHBA proteins exhibits considerable variability, meaning that single vaccines targeting NHBA often cannot cover a wide range of strains. The NHBA gene encodes approximately 420-500 amino acid residues. Besides variations at single or multiple sites, different NHBA proteins also exhibit insertions or deletions of single amino acids or short peptides. The amino acid homology between different NHBA proteins can be as low as 60%.

[0030] There are no particular limitations on the NHBA that can be used in this invention, and it can be any wild-type or mutant NHBA variant protein. Preferably, the amino acid sequence of the NHBA protein is as described in SEQ ID NO:5 (NHBA-peptide 20) or SEQ ID NO:6 (NHBA-peptide 669), and may also include amino acid sequences in which one or more amino acids are added, deleted, or substituted in SEQ ID NO:5 or SEQ ID NO:6.

[0031] The term "antigen" refers to a molecule or substance that can induce a specific immune response, such as the recombinant protein described in this invention.

[0032] The term "recombinant protein" refers to a first protein or peptide linked to a second protein or peptide (which may also contain a third protein or peptide) by peptide bonds between their respective N- and C-terminal amino acid residues, or by inserting two peptide bonds at the N- and C-termini of the first protein or peptide into an internal region of the second protein or peptide (which may also contain a third protein or peptide). A peptide bond is a covalent chemical bond formed between the carboxyl group of one amino acid and the amino group of another amino acid. As used herein, "recombinant protein" is generated by expressing a recombinant protein gene in an expression host, wherein the coding sequence of the first protein or peptide is linked to the coding sequence of the second protein or peptide (which may also contain a third protein or peptide).

[0033] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Nevertheless, this invention still intends to provide a more detailed description and explanation of these terms and phrases. In the event of any inconsistency between the terms and phrases mentioned and their known meanings, the meanings expressed in this invention shall prevail.

[0034] Example 1: Design of Recombinant Proteins

[0035] In the previous screening of this study, three highly immunogenic proteins were obtained: MenB-14 (FHbp-V231, amino acid sequence SEQ ID NO:7, of which amino acids 28-274 are V1 region (amino acid sequence SEQ ID NO:1), amino acids 282-536 are V2 region (amino acid sequence SEQ ID NO:2, amino acids 543-791 are V3 region (amino acid sequence SEQ ID NO:3), MenB-18 (NHBA-peptide 20, amino acid sequence SEQ ID NO:5, of which amino acids 25-427 are its domain portion) and MenB-23 (NHBA-peptide 669-GNA1030, amino acid sequence SEQ ID NO:8; of which the amino acid sequence of NHBA-peptide 669 region is SEQ ID NO:6).

[0036] Sixteen recombinant proteins were constructed by selecting FHbp (V1, V2, V3 regions), NHBA (peptide 20 and peptide 669), and GNA2091 antigen components (amino acid sequence SEQ ID NO:4) through domain recombination.

[0037] Recombinant proteins are classified into the following four categories based on their structural characteristics: Figure 1 ): FHbp recombinant protein ( Figure 1 a) FHbp-GNA2091 recombinant protein (1b) FHbp-NHBA recombinant protein (1c) NHBA recombinant protein ( Figure 1d). These are named MenB-23 (amino acid sequence SEQ ID NO:8), MenB-29 (amino acid sequence SEQ ID NO:9), MenB-30 (amino acid sequence SEQ ID NO:10), MenB-31 (amino acid sequence SEQ ID NO:11), MenB-32 (amino acid sequence SEQ ID NO:12), MenB-33 (amino acid sequence SEQ ID NO:13), MenB-34 (amino acid sequence SEQ ID NO:14), MenB-35 (amino acid sequence SEQ ID NO:15), MenB-36 (amino acid sequence SEQ ID NO:16), MenB-37 (amino acid sequence SEQ ID NO:17), MenB-38 (amino acid sequence SEQ ID NO:18), MenB-39 (amino acid sequence SEQ ID NO:19), MenB-24 (amino acid sequence SEQ ID NO:20), MenB-25 (amino acid sequence SEQ ID NO:21), and MenB-26 (amino acid sequence SEQ ID NO:17). NO:22), MenB-27 (amino acid sequence SEQ ID NO:23), and MenB-28 (amino acid sequence SEQ ID NO:24).

[0038] Example 2: Construction of recombinant protein expression plasmid

[0039] The target sequence is split and primers that can form overlap are designed. After the primers are mixed, the target fragment is amplified by PCR. The target fragment is then ligated into the corresponding vector. Positive clones with 100% correct sequence are selected and plasmids are extracted.

[0040] Experimental materials include primers, empty vector plasmid pET-30a(+), high-fidelity PCR polymerase (PV2), recombinase (Smart assemble), gel extraction kit (Axygen), and other routine molecular cloning reagents and consumables.

[0041] Taking the MenB-24 expression plasmid (SEQ ID NO:25) as an example, the construction process is described below:

[0042] Primer design and amplification: Overlap primers as shown in SEQ ID NO:26-31 were designed and synthesized according to the target sequence, and full-length PCR amplification was performed according to the reaction system and conditions shown in Tables 1-4.

[0043] Table 1. First-round full-length PCT reaction system

[0044]

[0045] Table 2. Conditions for the first round of full-length PCT reaction

[0046]

[0047] Table 3. Second-round full-length PCR reaction system

[0048]

[0049] Table 4. Conditions for the second round of full-length PCR reaction

[0050] Gel extraction and recovery of PCR amplification products: After the second round of PCR amplification, the PCR products are analyzed by electrophoresis. The PCR product image is in lane 2 to verify the product bands. The product bands must be unique and clear to meet the requirements. The PCR products that meet the requirements are then extracted from the gel (refer to the Axygen product manual). The recovered PCR products are used for the next ligation experiment.

[0051] Vector plasmid digestion: The digestion reaction was carried out at 37℃ for 60 min as shown in Table 5.

[0052] Table 5. Enzyme digestion system

[0053]

[0054] Recombination ligation experiment: Following the ligation system shown in Table 6, the cells were incubated at 52°C for 40 min, followed by transformation and a 5 min cooling period. The ligation product was then transferred into competent cells using standard methods, and single colonies were picked from overnight plates for identification according to the reaction system and conditions shown in Tables 7-8.

[0055] Table 6. Connection System

[0056]

[0057] Table 7. Colony PCR Reaction System

[0058] Table 8. Colony PCR reaction conditions

[0059] Plasmid extraction and sequencing: Positive clone bacterial cultures were cultured overnight, then plasmids were extracted and sequenced using forward sequencing primers, reverse sequencing primers, and walking sequencing primers to ensure the full length of the target sequence was detected. The sequencing primer sequences are shown in seq id no. 32-36.

[0060] Obtaining the correct plasmid: The final plasmid digestion bands were correct, and the sequencing results showed that the sequence was 100% correct.

[0061] Example 3: Construction of recombinant protein expression strains

[0062] The recombinant protein expression plasmid constructed in Example 2 was transformed into BL21(DE3) competent cells (purchased from Sangon Biotech Co., Ltd., catalog number: B528414), plated on LB solid medium containing kanamycin resistance, and colonies were picked and transferred to liquid LB medium. The cells were cultured at 37°C and 200 rpm. When the OD value of the bacterial culture reached 0.6-0.8, IPTG (isopropyl-β-D-thiogalactoside, purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number: ST098) was added to a final concentration of 1 mM to induce expression. After culturing at 20°C and 160 rpm for 16 h, the bacterial cells were collected by centrifugation, resuspended in PBS solution, and subjected to SDS-PAGE protein electrophoresis and Western blotting to identify the expression of bacterial proteins.

[0063] Example 4: Expression, purification, and concentration determination of recombinant protein

[0064] The bacterial precipitate collected by centrifugation in Example 3 was high-pressure disrupted. After high-pressure disruption, the sample was centrifuged at 7000 rpm for 90 min, and the supernatant was collected. The recombinant protein was purified by nickel column affinity chromatography. After purification, the protein sample with higher purity in the elution fraction was selected by SDS-PAGE electrophoresis and concentrated. Then, the concentration of the recombinant protein was determined according to the BCA method.

[0065] Example 5: Preparation of Recombinant Protein Vaccine

[0066] In a clean bench, the recombinant protein and adjuvant Al(OH)3 were mixed in PBS to a final concentration of 20µg recombinant protein + 200µg Al(OH)3 / 100µL vaccine. The mixture was placed on a shaker (150rpm, room temperature, 1h) to allow the recombinant protein and adjuvant to be fully adsorbed, thereby enhancing the immune response.

[0067] Example 6: Mouse Immunization Experiment

[0068] The steps for the immunoassay are as follows:

[0069] (1) 100 female BALB / C mice aged 6-8 weeks were selected and divided into 20 groups of 5 mice each.

[0070] (2) Immunization groups are shown in Table 9. Mice in groups 1-19 were immunized with 19 recombinant protein vaccines, and mice in group 20 served as the negative control group. The vaccine injection volume was 100µL / mouse.

[0071] (3) All injections were administered via intramuscular injection.

[0072] (4) Serum samples were obtained by collecting blood from the orbital venous plexus 3 weeks after the initial immunization (day 21) and 2 weeks after the second immunization (day 35). The serum was separated and frozen at -80°C for subsequent experiments.

[0073] Table 9. Immunization grouping of mice

[0074]

[0075] Example 7: ELISA detection of binding antibody titer

[0076] The antibody IgG titer of immune serum was determined using an ELISA method. The purified recombinant protein was diluted to 2 µg / mL, and 100 µL / well was added to each well of an ELISA plate. The plate was incubated overnight at 4°C. After washing and blocking, serially diluted mouse serum was added to the ELISA plate and incubated at 37°C for 1 hour. After washing, HRP-labeled goat anti-mouse IgG enzyme-labeled antibody (purchased from ZEN BIO, catalog number: 511103) was added and incubated at 37°C for 1 hour. After washing, TMB was added for color development for 2 minutes, and then stop solution (2M H₂SO₄, 50 µL / well) was added to terminate the reaction. The absorbance was measured using a dual-wavelength spectrophotometer: the dominant wavelength was 450 nm, and the reference wavelength was 630 nm. The corrected absorbance value (ΔOD = OD₄₅₀ - OD₆₃₀) was obtained by subtracting the corresponding 630 nm absorbance value (OD₄₅₀) from the 450 nm absorbance value (OD₄₅₀) of each well. Calculate the arithmetic mean of the corrected absorbance for each group. Using the serum from the non-immunized group as a negative control, the cut-off value is set at 2.1 times the mean ΔOD value of the negative control serum to determine the negative / positive reaction. The highest dilution with a positive reaction is the antibody titer of the sample to be tested, and the geometric mean titer (GMT) of each group of samples is calculated.

[0077] As shown in Table 10-11, specific antibodies were detected in the serum of mice in all groups 3 weeks after the initial immunization, but the titer of IgG antibodies was low. Two weeks after the second immunization, serum IgG antibody titers significantly increased. In summary, all 19 antigens could induce the production of binding antibodies effectively. Among them, MenB-14 (FHbp-V231) and MenB-23 (NHBA-peptide 669-GNA1030) induced higher antibody titers, suggesting that their immunogenicity was superior to that of the other antigens.

[0078] Table 10. Comparison of serum antigen-specific IgG antibody titers 3 weeks (day 21) after primary immunization with 19 recombinant protein vaccines

[0079]

[0080] Table 11. Comparison of serum antigen-specific IgG antibody titers 2 weeks (day 21) after secondary immunization with 19 antigens

[0081]

[0082] Example 8: Detection of bactericidal antibody titer

[0083] The testing steps are as follows:

[0084] (1) Strain resuscitation: The FHbp-V1 strain was taken out from the -80℃ freezer and spread on chocolate agar plates (purchased from Huankai Microbial Co., Ltd., catalog number: 029031) and placed in Incubate overnight (16-18 h) in a 37℃ constant temperature incubator.

[0085] (2) Bacterial subculture: Take the chocolate agar plate from step 1, pick 3-5 uniform single colonies, resuspend them in 100µL phosphate buffer, spread them evenly on a new chocolate agar plate, and continue to place them in the culture medium. Incubate at 37℃ for 4 hours.

[0086] (3) Culture medium preparation: Melt the agar culture medium and place it in a 45℃ constant temperature water bath to balance the temperature for later use.

[0087] (4) Add 25 µL of phosphate buffer to each well of the 96-well titration plate. Add 25 µL of the test serum (experimental group) or negative control serum (mouse serum immunized with PBS) to the first well of rows 1 to 6. Use a 2-fold serial dilution, with dilution factors of 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048, and 4096 respectively.

[0088] (5) 25 µL of rabbit complement (purchased from Zhongchuang Huike Co., Ltd., catalog number: ZC-PRO-001) was added to each well in the experimental group. Wells 1-6 in row 7 were set up as bacterial growth control: 25 µL of inactivated complement was added (water bath at 56℃ for 30 min) to verify the growth status of target bacteria; Wells 7-12 were set up as complement control: 25 µL of active complement was added to detect whether complement itself has bactericidal activity.

[0089] (6) Add 4 mL of phosphate buffer solution to the bacterial culture plate from step (2) that has been cultured for 4 h. Gently scrape off the bacterial growth and mix well. Measure the OD600 value of the bacterial suspension and adjust it to OD600 = 0.5 with phosphate buffer solution. Add 25 µL of the adjusted bacterial suspension to each well, shake well on a shaker for 5 min, and then incubate at 37 °C for 50 min.

[0090] (7) Take out the titration plate, add 100µL of 50% glucose injection and 100µL of 1% triphenyltetrazolium chloride (TTC) to each 10 mL agar medium, and add 50 μL of mixed medium to each well while shaking on the shaker.

[0091] (8) Place the titration plate in 5% Incubate at 37℃ for 18 h.

[0092] (9) Result determination:

[0093] Control well verification: The bacterial growth control, complement control, and negative control wells should all show evenly distributed red dot-like colonies. If the control wells do not meet expectations, the experiment needs to be repeated.

[0094] Bactericidal activity assessment: A well with significantly fewer colonies than the control well (≤50%) is considered positive (sterilized). A well with a colony count similar to the control well (>50%) is considered negative (not sterilized).

[0095] Titer calculation: The highest dilution of serum showing bactericidal positivity was used as the bactericidal antibody titer. The geometric mean titer (GMT) of each group of samples was calculated.

[0096] As shown in Tables 12-13, the results of the tests 3 weeks after the initial immunization (Table 12) showed that only the MenB-37 and MenB-35 groups of mice exhibited low bactericidal activity (GMT<16), while no bactericidal effect was detected in the other experimental groups (GMT≤4). However, the results of the tests 2 weeks after the secondary immunization (Table 13) showed that the bactericidal activity of the serum in all experimental groups was enhanced. Among them, the MenB-35 group (GNA2091-FHbp-V123 fusion antigen) showed the strongest immunogenicity, with a GMT of 256, which was significantly higher than that of other antigen groups.

[0097] Table 12. Comparison of serum bactericidal antibody titers 3 weeks (day 21) after primary immunization with 19 antigens

[0098] Note: Data in the table are sorted in descending order; "-" indicates no bactericidal effect.

[0099] Table 13. Comparison of serum bactericidal antibody titers 2 weeks (day 35) after secondary immunization with 19 antigens

[0100]

[0101] Note: Data in the table are sorted in descending order; "-" indicates no bactericidal effect.

[0102] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

[0103] The relevant sequences of this invention are as follows:

[0104] > FHbp-V1 region amino acid sequence

[0105] VAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRRFRI GDIAGEHTSFDKLPEGGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGKAQEVAGSAEVETANGIHHIGLAAKQ

[0106] SEQ ID NO:1

[0107] > FHbp-V2 region amino acid sequence

[0108] VAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFL VSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ

[0109] SEQ ID NO:2

[0110] > FHbp-V3 region amino acid sequence

[0111] SGSGGVAADIGTGLADALTTPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ

[0112] SEQ ID NO:3

[0113] > Amino acid sequence of GNA2091 antigen

[0114] MKPKPHTVRTLIAAIFSLALSGCVSAVIGSAAVGAKSAVDRRTTGAQTDDNVMALRIETTARSYLRQNNQTKGYTPQISVVGYNRHLLLLGQVATEGEKQFVGQIARSEQAAEGVYNYITVASLPRTAGDIAGDTWNTSKVRATLLGISPATQARVKIVTYGNVTYVMGILTPEEQAQITQKVSTTVGVQKVITLYQNYVQR

[0115] SEQ ID NO:4

[0116] > Amino acid sequence of NHBA-peptide 20

[0117] MFERSVIAMACIFALSACGGGGGGSPDVKSADTLSKPAAPVVAEKETEVKEDAPQAGSQGQGAPSTQGSQDMAAVSAENTGNGGAATTDKPKNEDEGPQNDMPQNSAESANQTGNNQPADSSDSAPASNPAPANGGSNFGRVDLANGVLIDGPSQNITLTHCKGDSCNGDNLLDEEAPSKSEFENLNESERIEKYKKDGKSDKFTNLVATAVQANGTNKYVIIYKDKSASSSSARFRRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGNYRYLTYGAEKLPGGSYALRVQGEPAKGEMLAGTAVYNGEVLHFHTENGRPYPTRGRFAAKVDFGSKSVDGIIDSGDDLHMGTQKFKAAIDGNGFKGTWTENGGGDVSGRFYGPAGEEVAGKYSYRPTDAEKGGFGVFAGKKEQDHHHHHHHH

[0118] SEQ ID NO:5

[0119] > Amino acid sequence of the 669 region of the NHBA-peptide

[0120] SPDVKSADTPSKPAAPVVAEKETEAKEDAPQAGSQGQDVPSTQGSQDMAAVSAENTGNGGAATADNPKNEDEVAQNDMPQNAAGTDSSTPNHTPDPNMLAGNMENQATDAGESSQPANQPDMANAADGMQGDDPSAGRQNAGNTAAQGANQAGNNQAAGSSDPIPASNPATTNSGGDFGRVDLANGIKLDGGSENVTLTHCKDKVCGSNFLDEEAPSKSEFEKLSDAEKINKYKKNGGKFTGLVATRVENNGLNQYTIIYQAQPTRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGNYRYLTYGAEKLPGGSYALSVQGKPAKGEMLAGAAVYNGEVLHFHTENGRPYPTRGRFAAKVDFGSKSVDGIIDSGDDLHMGTQKFKAAIDGNGFKGTWTENGGGDVSGKFYGPAGEEVAGKYSYRPTDAEKGGFGVFAGKKEQD

[0121] SEQ ID NO:6

[0122] > Amino acid sequence of MenB-14 (FHbp-V231)

[0123] MKATKLVLGAVILGSTLLAGCSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQCSSGGGGSGSGGVAADIGTGLADALTTPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQCSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRRFRIGDIAGEHTSFDKLPEGGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGKAQEVAGSAEVETANGIHHIGLAAKQHHHHHHHH

[0124] SEQ ID NO:7

[0125] > Amino acid sequence of MenB-23 (NHBA-peptide 669-GNA1030)

[0126] MFERSVIAMACIFALSACGGGGGGSPDVKSADTPSKPAAPVVAEKETEAKEDAPQAGSQGQDVPSTQGSQDMAAVSAENTGNGGAATADNPKNEDEVAQNDMPQNAAGTDSSTPNHTPDPNMLAGNMENQATDAGESSQPANQPDMANAADGMQGDDPSAGRQNAGNTAAQGANQAGNNQAAGSSDPIPASNPATTNSGGDFGRVDLANGIKLDGGSENVTLTHCKDKVCGSNFLDEEAPSKSEFEKLSDAEKINKYKKNGGKFTGLVATRVENNGLNQYTIIYQAQPTRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGNYRYLTYGAEKLPGGSYALSVQGKPAKGEMLAGAAVYNGEVLHFHTENGRPYPTRGRFAAKVDFGSKSVDGIIDSGDDLHMGTQKFKAAIDGNGFKGTWTENGGGDVSGKFYGPAGEEVAGKYSYRPTDAEKGGFGVFAGKKEQD GSGGGG ATYKVDEYHANARFAIDHFNTSTNVGGFYGLTGSVEFDQAKRDGKIDITIPIANLQSGSQHFTDHLKSADIFDAAQYPDIRFVSTKFNFNGKKLVSVDGNLTMHGKTAPVKLKAEKFNCYQSPMEKTEVCGGDFSTTIDRTKWGMDYLVNVGMTKSVRIDIQIEAAKQHHHHHHHH

[0127] SEQ ID NO:8

[0128] > Amino acid sequence of MenB-29

[0129] MKATKLVLGAVILGSTLLAGCSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRRFRIGDIAGEHTSFDKLPEGGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGKAQEVAGSAEVETANGIHHIGLAAKQCSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQCSSGGGGSGSGGVAADIGTGLADALTTPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQHHHHHHHH

[0130] SEQ ID NO:9

[0131] > Amino acid sequence of MenB - 30

[0132] MKATKLVLGAVILGSTLLAGCSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRRFRIGDIAGEHTSFDKLPEGGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGKAQEVAGSAEVETANGIHHIGLAAKQCSSGGGGSGSGGVAADIGTGLADALTTPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQCSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQHHHHHHHH

[0133] SEQ ID NO:10

[0134] > Amino acid sequence of MenB-31

[0135] MKATKLVLGAVILGSTLLAGCSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQCSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRRFRIGDIAGEHTSFDKLPEGGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGKAQEVAGSAEVETANGIHHIGLAAKQCSSGGGGSGSGGVAADIGTGLADALTTPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQHHHHHHHH

[0136] SEQ ID NO:11

[0137] > Amino acid sequence of MenB - 32

[0138] MKATKLVLGAVILGSTLLAGCSSGGGGSGSGGVAADIGTGLADALTTPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQCSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRRFRIGDIAGEHTSFDKLPEGGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGKAQEVAGSAEVETANGIHHIGLAAKQCSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQHHHHHHHH

[0139] SEQ ID NO:12

[0140] > Amino acid sequence of MenB-33

[0141] MKATKLVLGAVILGSTLLAGCSSGGGGSGSGGVAADIGTGLADALTTPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQCSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQCSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRRFRIGDIAGEHTSFDKLPEGGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGKAQEVAGSAEVETANGIHHIGLAAKQHHHHHHHH

[0142] SEQ ID NO:13

[0143] > Amino acid sequence of MenB - 34

[0144] MKPKPHTVRTLIAAIFSLALSGCVSAVIGSAAVGAKSAVDRRTTGAQTDDNVMALRIETTARSYLRQNNQTKGYTPQISVVGYNRHLLLLGQVATEGEKQFVGQIARSEQAAEGVYNYITVASLPRTAGDIAGDTWNTSKVRATLLGISPATQARVKIVTYGNVTYVMGILTPEEQAQITQKVSTTVGVQKVITLYQNYVQRCSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQCSSSGGGSGSGGVAADIGTGLADALTTPLDHKDK GLKSLTLEDSIPQNGTLTSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQCSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRRFRIGDIAGEHTSFDKLPEGGSATYRGTAFGSSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGKAQEVAGSAEVETANGIHHIGLAAKQHHHHHHHH

[0145] SEQ ID NO:14

[0146] MenB-35 amino acid sequence

[0147] MKPKPHTVRTLIAAIFSLALSGCVSAVIGSAAVGAKSAVDRRTTGAQTDDNVMALRIETTARSYLRQNNQTKGYTPQISVVGYNRHLLLLGQVATEGEKQFVGQIARSEQAAEGVYNYITVASLPRTAGDIAGDTWNTSKVRATLLGISPATQARVKIVTYGNVTYVMGILTPEEQAQITQKVSTTVGVQKVITLYQNYVQRCSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRRFRIGDIAGEHTSFDKLPEGGSATYRGTAFGSDDAGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGKAQEVAGSAEVETANGIHHIGLAAKQCSSGGGGVAADIGAGLADALTAPLDHKDKSLQS LTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQCSSGGGSGSGGVAADIGTGLADALTTPLDHKDKGLKLSLTLEDSIPQNGTLTSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQHHHHHHHH

[0148] SEQ ID NO:15

[0149] MenB-36 amino acid sequence

[0150] MKPKPHTVRTLIAAIFSLALSGCVSAVIGSAAVGAKSAVDRRTTGAQTDDNVMALRIETTARSYLRQNNQTKGYTPQISVVGYNRHLLLLGQVATEGEKQFVGQIARSEQAAEGVYNYITVASLPRTAGDIAGDTWNTSKVRATLLGISPATQARVKIVTYGNVTYVMGILTPEEQAQITQKVSTTVGVQKVITLYQNYVQRCSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRRFRIGDIAGEHTSFDKLPEGGSATYRGTAFGSDDAGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGKAQEVAGSAEVETANGIHHIGLAAKQCSSSGGGSGSGGVAADIGTGLADALTTPLDHKD KGLKSLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQCSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQHHHHHHHH

[0151] SEQ ID NO:16

[0152] MenB-37 amino acid sequence

[0153] MKPKPHTVRTLIAAIFSLALSGCVSAVIGSAAVGAKSAVDRRTTGAQTDDNVMALRIETTARSYLRQNNQTKGYTPQISVVGYNRHLLLLGQVATEGEKQFVGQIARSEQAAEGVYNYITVASLPRTAGDIAGDTWNTSKVRATLLGISPATQARVKIVTYGNVTYVMGILTPEEQAQITQKVSTTVGVQKVITLYQNYVQRCSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQCSSGGGGVAADIGAGLADALTAPLDHKDKGLQSL TLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRRFRIGDIAGEHTSFDKLPEGGSATYRGTAFGSDDAGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGKAQEVAGSAEVETANGIHHIGLAAKQCSSGGGSGSGGVAADIGTGLADALTTPLDHKDKGLKSLTLEDSIPQNGTLTSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSDDAGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQHHHHHHHH

[0154] SEQ ID NO:17

[0155] MenB-38 amino acid sequence

[0156] MKPKPHTVRTLIAAIFSLALSGCVSAVIGSAAVGAKSAVDRRTTGAQTDDNVMALRIETTARSYLRQNNQTKGYTPQISVVGYNRHLLLLGQVATEGEKQFVGQIARSEQAAEGVYNYITVASLPRTAGDIAGDTWNTSKVRATLLGISPATQARVKIVTYGNVTYVMGILTPEEQAQITQKVSTTVGVQKVITLYQNYVQRCSSGGGGSGSGGVAADIGTGLADALTTPLDHKDKGLKSLTLEDSIPQNGTLTSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQCSSGGGGVAADIGAGLADALTAPLDH KDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRRFRIGDIAGEHTSFDKLPEGGSATYRGTAFGSDDAGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGKAQEVAGSAEVETANGIHHIGLAAKQCSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSDDAGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQHHHHHHHH

[0157] SEQ ID NO:18

[0158] MenB-39 amino acid sequence

[0159] MKPKPHTVRTLIAAIFSLALSGCVSAVIGSAAVGAKSAVDRRTTGAQTDDNVMALRIETTARSYLRQNNQTKGYTPQISVVGYNRHLLLLGQVATEGEKQFVGQIARSEQAAEGVYNYITVASLPRTAGDIAGDTWNTSKVRATLLGISPATQARVKIVTYGNVTYVMGILTPEEQAQITQKVSTTVGVQKVITLYQNYVQRCSSGGGGSGSGGVAADIGTGLADALTTPLDHKDKGLKSLTLEDSIPQNGTLTSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQCSSGGGGVAADIGAGLADALTAPLDH KDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQCSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRRFRIGDIAGEHTSFDKLPEGGSATYRGTAFGSSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGKAQEVAGSAEVETANGIHHIGLAAKQHHHHHHHH

[0160] SEQ ID NO:19

[0161] MenB-24 amino acid sequence

[0162]

[0163] SEQ ID NO:20

[0164] MenB-25 amino acid sequence

[0165]

[0166] SEQ ID NO:21

[0167] MenB-26 amino acid sequence

[0168]

[0169] SEQ ID NO:22

[0170] MenB-27 amino acid sequence

[0171]

[0172] SEQ ID NO:23

[0173] > Amino acid sequence of MenB-28

[0174] MFERSVIAMACIFALSACGGGGGGSPDVKSADTLSKPAAPVVAEKETEVKEDAPQAGSQGQGAPSTQGSQDMAAVSAENTGNGGAATTDKPKNEDEGPQNDMPQNSAESANQTGNNQPADSSDSAPASNPAPANGGSNFGRVDLANGVLIDGPSQNITLTHCKGDSCNGDNLLDEEAPSKSEFENLNESERIEKYKKDGKSDKFTNLVATAVQANGTNKYVIIYKDKSASSSSARFRRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGNYRYLTYGAEKLPGGSYALRVQGEPAKGEMLAGTAVYNGEVLHFHTENGRPYPTRGRFAAKVDFGSKSVDGIIDSGDDLHMGTQKFKAAIDGNGFKGTWTENGGGDVSGRFYGPAGEEVAGKYSYRPTDAEKGGFGVFAGKKEQDGGGGGGSPDVKSADTPSKPAAPVVAEKETEAKEDAPQAGSQGQDVPSTQGSQDMAAVSAENTGNGGAATADNPKNEDEVAQNDMPQNAAGTDSSTPNHTPDPNMLAGNMENQATDAGESSQPANQPDMANAADGMQGDDPSAGRQNAGNTAAQGANQAGNNQAAGSSDPIPASNPATTNSGGDFGRVDLANGIKLDGGSENVTLTHCKDKVCGSNFLDEEAPSKSEFEKLSDAEKINKYKKNGGKFTGLVATRVENNGLNQYTIIYQAQPTRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGNYRYLTYGAEKLPGGSYALSVQGKPAKGEMLAGAAVYNGEVLHFHTENGRPYPTRGRFAAKVDFGSKSVDGIIDSGDDLHMGTQKFKAAIDGNGFKGTWTENGGGDVSGKFYGPAGEEVAGKYSYRPTDAEKGGFGVFAGKKEQDHHHHHHHH

[0175] SEQ ID NO:24

[0176] MenB-29 nucleotide sequence

[0177]

[0178] SEQ ID NO:25

[0179] > G62338-9FGTTTAACTTTAAGAAGGAGATATAcatATGAAGGCAACCAAACTGGTGCTGGGTGCCGTTATTC

[0180] SEQ ID NO:26

[0181] > G62338-9f1CTGGTGCTGGGTGCCGTTATTCTGGGCAGTACCCTGCTGGCCGGTTGCAGTAGTGGTGGCGGTGGTAG

[0182] SEQ ID NO:27

[0183] > G62338-9r1CGGCAACGCCACCGCCACCACTGCTACACTGTTTTGCTGCCAGACCAATATGATG

[0184] SEQ ID NO:28

[0185] > G62338-9f2CATCATATTGGTCTGGCAGCAAAACAGTGTAGCAGTGGTGGCGGTGGCGTTGCCG

[0186] SEQ ID NO:29

[0187] > G62338-9r2CGGAGCTCGAATTCGGATCCCTATTAGTGGTGGTGGTGGTGGTGGTGGTGCTGTTTACCGGCAATGCCGAT

[0188] SEQ ID NO:30

[0189] > G62338-9RCCGCAAGCTTGTCGACGGAGCTCGAATTCGGATCCCTA

[0190] SEQ ID NO:31

[0191] > pET-seqF

[0192] GGCCCCAAGGGGTTATGCTAGT

[0193] SEQ ID NO:32

[0194] > pET-seqR

[0195] GATCCCGCGAAATTAATACG

[0196] SEQ ID NO:33

[0197] > G62338-9-seq1

[0198] GTAAAGCCTTTAGCAGCGAT

[0199] SEQ ID NO:34

[0200] G62338-9-seq2

[0201] ATAAACAGAGCCATAGTGCA

[0202] SEQ ID NO:35

[0203] G62338-9-seq3

[0204] GATAAAGTTAGCCGCTTTGA

[0205] SEQ ID NO:36

[0206] MenB-24 gene sequence

[0207]

[0208] SEQ ID NO:37

[0209] Full sequence of MenB-24 expression plasmid

[0210]

[0211] SEQ ID NO:38。

Claims

1. A recombinant protein, characterized in that, It includes Factor H Binding Protein Variant 1 (FHbp V1 variant protein), Factor H Binding Protein Variant 2 (FHbp V2 variant protein), and Factor H Binding Protein Variant 3 (FHbp V3 variant protein), wherein the amino acid sequence of Factor H Binding Protein Variant 1 is shown in SEQ ID NO: 1, the amino acid sequence of Factor H Binding Protein Variant 2 is shown in SEQ ID NO: 2, and the amino acid sequence of Factor H Binding Protein Variant 3 is shown in SEQ ID NO:

3.

2. The recombinant protein of claim 1, further comprising GNA2091 or Neisserial Heparin Binding Antigen (NHBA protein), wherein the amino acid sequence of GNA2091 is shown in SEQ ID NO: 4, and the amino acid sequence of the heparin binding protein is shown in positions 25-427 of SEQ ID NO: 5, or as shown in SEQ ID NO:

6.

3. The recombinant protein according to any one of claims 1-2, further comprising a signal peptide and / or a linker peptide.

4. The recombinant protein according to any one of claims 1-3, having an amino acid sequence as described in any one of SEQ ID NO: 7, 9-23.

5. A nucleic acid molecule, characterized in that, Its encoding is the recombinant protein as described in any one of claims 1-4.

6. A recombinant expression vector, characterized in that, It contains the nucleic acid molecule as described in claim 5.

7. A host cell, characterized in that, It expresses the fusion protein as described in any one of claims 1-4; or contains the nucleic acid molecule as described in claim 5; or contains the recombinant expression vector as described in claim 6.

8. A method for preparing recombinant proteins, characterized in that, The method includes: Culture medium is obtained by culturing the host cells as described in claim 7; and the recombinant protein is isolated from the culture medium.

9. A vaccine composition, characterized in that, It includes the recombinant protein as described in any one of claims 1-4, the nucleic acid molecule as described in claim 5, or the recombinant expression vector as described in claim 6; And a vaccine-acceptable vector; The vaccinologically acceptable carriers include aluminum adjuvant, MF59, Freund's complete adjuvant, Freund's incomplete adjuvant, CpG adjuvant, BCG ribonucleic acid, or monophospholipase A.

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