NHBA recombinant protein

Abstract

According to the invention, a group B meningococcus NHBA protein is artificially modified to obtain a P4-NHBA recombinant protein, and the P4-NHBA recombinant protein is used as an antigen to prepare a group B meningococcus vaccine. Experiments prove that the recombinant protein can be efficiently expressed and modified through an escherichia coli expression system, has good immunogenicity and can be used for preparing a vaccine composition for preventing group B meningococcus.

Description

Technical Field

[0001] The present invention relates to the field of biological products, and more specifically, to the field of meningococcal vaccines, in particular to recombinant proteins and immunogenic compositions related to group B meningococci, as well as their uses and preparation methods. Background Art

[0002] Neisseria meningitidis, also known as Neisseria meningitidis, meningococcus, or simply meningococcus, is a Gram-negative coccus confined to humans. It belongs to the family Neisseriaceae and is diplococcoid. It is a major pathogen causing epidemic meningitis and sepsis worldwide. Neisseria meningitidis is a Gram-negative coccus confined to humans. It belongs to the family Neisseriaceae and is diplococcoid. Therefore, it is also called meningococcus or meningococcus. It is a major pathogen causing epidemic meningitis and bacteremia worldwide. Meningococci can be encapsulated or non-capsulated. Their genome consists of approximately 2 million base pairs, containing approximately 2,000 coding genes. Based on the different capsular polysaccharide structures, meningococci can be divided into at least 13 serogroups, but only infection with 6 of them (A, B, C, W135, X and Y) can cause more serious diseases.

[0003] Currently, most countries and regions in the world have marketed polysaccharide or polysaccharide conjugate vaccines for the prevention of epidemic meningitis caused by groups A, C, W-135, and Y. In July 2023, the pentavalent polysaccharide conjugate vaccine MenFive, jointly developed by the Serum Institute of India and the PATH Foundation, targeting groups A, C, W, X, and Y, was launched. TM The WHO has also given priority recommendation for vaccination of people aged 1-85 years. Due to the difficulty in developing a serogroup B meningococcal vaccine and its late release to the market, it is currently only available in certain countries and regions in Europe and the United States. There is no vaccine for serogroup B meningococci available in China. Furthermore, with the use of other types of meningococcal vaccines in most parts of the world, serogroup B meningococci has become the most prevalent meningococcal serogroup worldwide. The relative prevalence of serogroup B meningococci in China is also gradually increasing, making it the main pathogen causing meningitis. Therefore, there is an urgent need to develop a preventive vaccine against serogroup B meningococci.

[0004] Compared with the marketed ACYW135 group meningococcal vaccine, the research and development path of group B meningococcal vaccine is quite different and the research and development is also more difficult. The latter is mainly developed based on the surface capsular polysaccharide of meningococci, but this development strategy is not applicable to group B meningococcal vaccine. Early studies have shown that vaccines developed based on group B meningococcal capsular polysaccharides not only have low immunogenicity and are almost difficult to induce antibody responses, but also have the risk of inducing autoimmune reactions. Currently, only two group B meningococcal vaccines have been approved for marketing in Europe and the United States, namely Bexsero from GSK and Trumenba from Pfizer. No similar products have been approved for marketing in China. Both marketed vaccines are mainly based on the recombinant protein technology platform of the Escherichia coli prokaryotic expression system and use aluminum adjuvants to enhance immune response. GSK's Bexsero vaccine contains four antigen components, namely OMV (bacterial outer membrane vesicles), NadA (Neisserial adhesin A) protein, NHBA (Neisseria Heparin Binding Antigen, also known as GNA2132) protein, and a variant of fHbp (factor H-binding protein, also known as GNA1870) protein (subfamily B); Pfizer's Trumenba vaccine only uses two different variants of fHbp protein (subfamily A and subfamily B) as antigens.

[0005] Although both vaccines have been approved for marketing, their antigenic design primarily targets serotype B meningococcal strains prevalent in Europe and the United States, potentially less responsive to serotype B meningococcal strains prevalent in China. For example, the OMV, one of the main components of the Bexsero vaccine, derives its primary antigenic protein from the PorA antigen protein on the bacterial surface. However, the PorA sequence is poorly conserved, resulting in poor broad-spectrum protection from the OMV vaccine. OMV-based vaccines, when used alone, are generally used primarily in localized settings. Furthermore, OMV-based vaccines offer poor immunity and offer poor protection in younger infants and young children. Another component of the Bexsero vaccine, the NadA protein, primarily mediates the adhesion of serotype B meningococci to nasal epithelial cells. However, molecular epidemiological analysis of serotype B meningococcal strains in China revealed that over 90% of strains do not contain the NadA gene (Zhu Bingqing et al., Chinese Journal of Preventive Medicine, 2019). The NHBA protein is one of the main antigenic components contained in the marketed Bexsero vaccine. The sequence of this protein is relatively conserved among globally prevalent meningococci, with no obvious subtype distinction. It is expressed to a certain extent on the surface of almost all group B meningococci and can therefore induce potential broad-spectrum antibodies. However, in order to develop a group B meningococcal vaccine suitable for the Chinese population, it is still necessary to study NHBA to further improve its immunogenicity. Summary of the Invention

[0006] In view of the above problems, the object of the present invention is to provide a P4-NHBA recombinant protein with improved immunogenicity, an immunogenic composition comprising the P4-NHBA recombinant protein, a nucleic acid encoding the P4-NHBA recombinant protein, an expression vector comprising the nucleic acid, an expression system cell comprising the expression vector protein, and uses of the recombinant protein and the immunogenic composition.

[0007] About P4-NHBA recombinant protein:

[0008] (1) Its structural characteristics from N-terminus to C-terminus are: P4-linker-NHBA; wherein ① P4 at the N-terminus represents the P4 lipoprotein signal peptide from nontypable Haemophilus influenzae (Green BA et al., INFECTION AND IMMUNITY, Sept. 1991, p. 3191-3198), referred to as P4 signal peptide (its sequence is shown in SEQ ID NO: 3); ② NHBA at the C-terminus represents NHBA recombinant protein, which consists of the amino acid sequence shown in SEQ ID NO: 1 (or the amino acid sequence of SEQ ID NO: 2). NO:1 is equal in length and has more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% identity) and the 1st to xth amino acids at the N-terminus are deleted; ③ The linker located between the two represents a connecting peptide, and its length is 0 to 15 amino acid residues (i.e., the length of the connecting peptide can be any integer between 0 and 15, including the end value). When the length is 0, it means that no connecting peptide is required, that is, the front and back elements are directly covalently connected in series. Among them, when the linker length is 0: x = 17; when the linker length is not 0: the N-terminus of the linker is cysteine, and 18≤x≤24 (i.e., the value of x can be any positive integer between 18 and 24, including the end value).

[0009] (2) Preferably, for NHBA, wherein x=17, that is, the NHBA is obtained by deleting the 1st to 17th amino acids at the N-terminus of SEQ ID NO:1 (wherein, the retained amino acid residues "CGGGGGG" at positions 18 to 24 are rich in glycine, which has a small molecular weight and flexibility and can act as a connecting peptide, so there is no need to add an additional connecting peptide), and its N-terminus is directly covalently connected in series with the C-terminus of the P4 signal peptide. The amino acid sequence of the resulting P4-NHBA recombinant protein is shown in SEQ ID NO:4.

[0010] (3) In the above preferred embodiment, the retained amino acid residues "CGGGGGG" at positions 18 to 24 of SEQ ID NO: 1 are equivalent to a connecting peptide. Based on the same underlying logic, when designing the sequence of NHBA, part or all of the amino acid residues at positions 18 to 24 of SEQ ID NO: 1 can also be removed, and an additional connecting peptide can be added. The N-terminus of the connecting peptide is Cys (as the lipidation site of the mature protein after the P4 signal peptide is removed), and the downstream can be selected from Gn (polyglycine), (GGGGS)n or (EAAAK)n, where n represents the number of repetitions. Those skilled in the art can reasonably expect that the obtained fusion protein can also achieve the same or similar technical effects as the preferred embodiment.

[0011] The present invention also provides an immunogenic composition comprising the P4-NHBA recombinant protein; preferably, the immunogenic composition further comprises an adjuvant, and more preferably, the adjuvant is an aluminum adjuvant, such as aluminum phosphate or aluminum hydroxide adjuvant, more preferably aluminum hydroxide adjuvant.

[0012] The present invention also provides a polynucleotide encoding the P4-NHBA recombinant protein. Preferably, the polynucleotide comprises SEQ ID NO: 6.

[0013] The present invention also provides the use of the P4-NHBA recombinant protein or an immunogenic composition comprising the same in the preparation of a medicament for inducing an immune response in a mammal, preventing or alleviating Neisseria meningitidis infection and / or infection caused by Neisseria meningitidis; preferably, the Neisseria meningitidis is group B Neisseria meningitidis.

[0014] Beneficial effects of the present invention:

[0015] (1) Immunogenicity is further enhanced by adding a P4 signal peptide sequence to the NHBA recombinant protein. The P4 signal peptide can further guide the recombinant protein to the bacterial cell membrane after translation and expression in E. coli. The signal peptide is then degraded and removed, while the N-terminal cysteine (Cys) of the mature antigen protein located on the membrane undergoes lipid modification. Experimental data of the present invention show that the P4-NHBA recombinant protein has improved immunogenicity compared to the NHBA recombinant protein.

[0016] (2) The present invention optimizes the codons of the nucleotide coding sequence of the recombinant protein, which is conducive to efficient expression in the Escherichia coli expression system; the designed recombinant protein antigen can obtain soluble protein after expression in Escherichia coli, and experiments such as electrophoresis and ELISA have proved that the structure, modification and function of the prepared recombinant protein meet the requirements, which is conducive to vaccine process amplification and process optimization. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : SDS-PAGE electrophoresis results of NHBA and P4-NHBA recombinant proteins.

[0018] Figure 2 : In vitro affinity ELISA results of different concentrations of NHBA and P4-NHBA recombinant proteins with heparin. DETAILED DESCRIPTION

[0019] Example 1: Construction and screening of antigenic proteins and engineered strains

[0020] (1) Construction of recombinant antigen protein

[0021] The overall sequence of the NHBA protein is highly conserved and has not been subtyped. The amino acid sequences of the NHBA proteins of 127 wild-type serogroup B meningococcal strains prevalent in China were retrieved from the PubMLST database. The NHBA sequence similarity between strains was high, ranging from 79.1% to 100%. The NHBA protein sequence of the wild-type strain "Nm510510" (shown in SEQ ID NO:1) was selected as the antigen sequence to construct recombinant proteins, resulting in recombinant NHBA antigen and recombinant P4-NHBA antigen.

[0022] Recombinant NHBA antigen (NHBA recombinant protein): Based on the amino acid sequence of the NHBA protein of the Nm510510 strain shown in SEQ ID NO: 1, the amino acid residues 1 to 24 at the N-terminus are removed and replaced with two amino acid residues "MA" to obtain the NHBA recombinant protein, the sequence of which is specifically shown in SEQ ID NO: 2.

[0023] Recombinant P4-NHBA antigen (P4-NHBA recombinant protein): Based on the amino acid sequence of the Nm510510 strain NHBA protein shown in SEQ ID NO: 1, amino acid residues 1 to 17 at the N-terminus were removed, and the P4 signal peptide sequence shown in SEQ ID NO: 3 was added to the N-terminus to obtain the P4-NHBA recombinant protein, the sequence of which is specifically shown in SEQ ID NO: 4.

[0024] In summary, the specific amino acid sequences involved in this embodiment are as follows:

[0025] SEQ ID NO: 1: amino acid sequence of NHBA protein of strain Nm510510;

[0026] SEQ ID NO: 2: NHBA recombinant protein amino acid sequence;

[0027] SEQ ID NO: 3: P4 signal peptide amino acid sequence;

[0028] SEQ ID NO: 4: P4-NHBA recombinant protein amino acid sequence.

[0029] (2) Construction of recombinant plasmid: After confirming all the antigen components and their amino acid sequences of the immune composition, codon optimization is performed according to the expression host being Escherichia coli, thereby obtaining a DNA sequence encoding the target antigen protein. By means of genetic engineering, the DNA sequence encoding the target antigen protein is artificially synthesized. After confirming that the sequence is correct by checking the sequencing report, the synthesized DNA sequence is respectively inserted between the NdeI and HindIII restriction sites in the expression plasmid pET-30a (+), thereby obtaining a recombinant expression plasmid that can express each antigen respectively, and further transformed into a cloning strain such as DH5α or TOP10 for amplification and culture of the recombinant expression plasmid. After amplification and culture, a certain amount of purified recombinant expression plasmid can be extracted using a plasmid extraction kit, and the concentration and purity of the recombinant plasmid are determined by NanoDrop, the recombinant plasmid is subjected to enzyme digestion detection by enzyme digestion map, or the target sequence is identified by direct sequencing. The aforementioned codon-optimized DNA sequence information is as follows:

[0030] SEQ ID NO: 5: DNA sequence encoding the aforementioned NHBA recombinant protein;

[0031] SEQ ID NO: 6: DNA sequence encoding the aforementioned P4-NHBA recombinant protein.

[0032] (III) Recombinant plasmid transformation: The recombinant plasmid whose identification results are consistent with the expected sequence design is transformed into Escherichia coli BL21 competent cells to construct the engineered strain. Take about 100 ng of recombinant plasmid DNA, add it to 50 μl of competent cells placed on ice, mix gently, incubate on ice for 30 minutes, and then heat shock in a water bath at 42°C for 30 seconds. Then quickly transfer the sample tube to ice and incubate for 2 minutes, add 250 μl of SOC medium, and culture on a shaker at 37°C, 220 rpm for 45 minutes to 1 hour. After completion, spread the bacterial liquid on the LB solid medium of the corresponding resistance and culture it in a constant temperature incubator at 37°C overnight.

[0033] (IV) Strain PCR identification: scrape 10-50 single colonies grown on the solid culture medium after overnight culture and dissolve them directly in about 10μl sterile water to make a bacterial solution. Take about 1μl of the bacterial solution as a template for colony PCR to identify the target gene. The colony PCR test process is as follows: G2 Hot Start Master Mixes (Promega) were used according to the instructions. The forward primer sequence was CGATGCGTCCGGCGTAGA; the reverse primer sequence was GCTAGTTATTGCTCAGCGG. Approximately 5-10 μl of the colony PCR product was electrophoresed on a 1% agarose gel at 100V for 50 minutes. Afterward, the amplified product was aligned with the gel to ensure that it conformed to the expected size. The remaining PCR product can be sequenced to further verify that the target sequence conformed to the expected size.

[0034] (V) Expression of recombinant protein in small amounts: Take an appropriate amount of the bacterial solution in the above sterile water and add it to about 2 ml of liquid LB medium. Incubate at 37°C, 220 rpm, and culture overnight. Inoculate the overnight cultured bacterial solution into 10 ml of LB liquid medium according to a certain ratio and culture at 37°C, 220 rpm until the OD 600 When the value is between 0.6 and 0.8, add IPTG to a final concentration of 0.5 mM and induce for 4 hours. Collect the induced bacterial suspension, centrifuge at 4000 g for 10 minutes at 4°C, discard the supernatant, and store the bacteria at -20°C.

[0035] (VI) SDS-PAGE Identification: Resuspend the cells collected in the previous step in buffer. Add 5× Loading Buffer to the resuspended cell suspension and mix thoroughly. After incubation at 100°C, load the sample and perform conventional SDS-PAGE analysis. Compare the samples before and after induction to see if a distinct, thick protein band appears near the target antigen size. If so, strain screening is successful. Select the clone with the thickest band as the selected high-expressing engineered strain.

[0036] Example 2: Large-scale expression of engineered strains

[0037] Take a small amount of the high-expression strain obtained by screening and save the bacterial liquid, streak it on LB solid medium plate, and culture it at 37℃ overnight. After culture, pick a single clone and inoculate it into LB liquid medium, and culture it at 37℃, 220rpm, and culture it overnight. Inoculate the overnight culture liquid into a larger volume of LB liquid medium according to a certain ratio, and culture it at 37℃, 220rpm until the OD 600 When the pH value is 0.6-0.8, add IPTG to a final concentration of 0.5-1 mM and induce at 37°C, 220 rpm for 4 hours. Centrifuge at 4000g, 4°C for about 10 minutes until the cells are completely settled. Discard the supernatant and store the fermented cells at -20°C.

[0038] Example 3: Extraction and purification of recombinant protein

[0039] Recombinant protein extraction: Remove and collect the cells, resuspend them in lysis buffer (10mM Tris-HCl, 100mM NaCl, 1mM EDTA), and use an ultrasonic disruptor or homogenizer to disrupt the cell suspension. Centrifuge to separate the cell debris precipitate and the cell lysis supernatant. Take a small amount of the cell debris precipitate and the cell lysis supernatant for SDS-PAGE target protein identification. If the target protein is present in large quantities in the cell lysis supernatant, aliquot and save the cell lysis supernatant for downstream purification; if the target protein is present in large quantities in the cell debris precipitate, add an appropriate amount of 10mM Tris-HCl buffer containing detergent to resuspend the precipitate, incubate at 4°C for 1-2 hours, incubate overnight, sonicate or homogenize, and extract the target protein into the resuspended supernatant to ensure that all proteins are present in a soluble form, which is convenient for downstream protein purification.

[0040] Recombinant protein purification: After cleaning the nickel column, equilibrate the nickel column with 20mM PB, 0.5M NaCl, 20mM imidazole (pH 7.4) buffer. Filter the supernatant containing the soluble target protein with a 0.22μm membrane and load it. Wash it with 5 column volumes of 20mMPB, 0.5M NaCl containing 50mM imidazole. Then elute it with 3 column volumes of 500mM imidazole, 20mM PB, 0.5M NaCl solution and collect the flow-through. Use SDS-PAGE electrophoresis to detect the purification effect of the target protein. The results are as follows: Figure 1 As shown, after recombinant protein expression and purification in an E. coli system, the desired protein product can be obtained with high purity. The eluted purified harvest is centrifuged through a 50 kDa ultrafiltration membrane, exchanged into the required buffer system, and concentrated to the desired concentration and volume. After sterilization and filtration through a 0.22 μm membrane, the recombinant protein stock solution is obtained.

[0041] Example 4: Evaluation of in vitro activity of purified recombinant protein

[0042] NHBA has an affinity for its host target protein and can bind to heparin in vitro. Therefore, the biological activity of the purified recombinant protein product can be tested by in vitro affinity binding ELISA, thereby preliminarily confirming the correct protein structure and biological function of the recombinant protein expression product.

[0043] NHBA-Heparin In Vitro Affinity ELISA: Add 100 μl of serially diluted recombinant NHBA protein (NHBA, P4-NHBA) to a specific concentration into a 96-well microtiter plate and coat overnight at 2-8°C. Discard the contents of the wells and wash the plate five times with approximately 200 μl of PBST buffer. Discard the contents. Add 150 μl of milk blocking solution to each well and block at room temperature for approximately 1 hour. Discard the blocking solution and wash the plate five times with approximately 200 μl of PBST buffer. Discard the contents. Add 100 μl of heparin-biotin solution diluted to 1 μg / ml to each well and incubate at room temperature for approximately 2 hours. Discard the contents of the wells and wash the plate five times with approximately 200 μl of PBST buffer. Discard the contents. Add approximately 100 μl of avidin-HRP to each well and incubate at room temperature for approximately 1 hour. After incubation, wash each well and add about 100 μl of chromogenic substrate TMB to each well. Color development is carried out for 10-15 minutes in the dark. Add about 100 μl of stop solution to each well and read the OD value with a microplate reader. 450mm -OD 570mm Absorbance value. The results are as follows Figure 2 The results are shown in Figure 2, which show in vitro affinity ELISA results for different recombinant NHBA proteins and heparin. The results demonstrate that the recombinant NHBA proteins constructed in the present invention, regardless of whether the protein is lipidated by the addition of a P4 signal peptide at the N-terminus, exhibit good in vitro affinity for heparin. This indicates that after soluble expression in E. coli, the recombinant proteins can fold into the correct protein structure and exhibit significant biological activity, potentially enabling further applications such as as antigens in vaccine development.

[0044] Example 5: Immunogenicity studies

[0045] Component preparation: The purified different recombinant protein components and aluminum hydroxide adjuvant were mixed and adsorbed separately to prepare an immune composition containing 100 μg / ml of recombinant protein and 80 μg / ml of aluminum adjuvant.

[0046] Animal Immunization: 6-8 week old female BALB / c mice were inoculated with the antigen prepared according to the previous examples, with 8-10 mice per group. Each group of mice was immunized three times intramuscularly on days 0, 21, and 42 at a dose of 100 μl per mouse. Blood was collected 14 days after the final immunization, and serum was isolated for antibody titer determination.

[0047] Serum separation: Incubate mouse peripheral blood at 37°C for 1 hour, then at 4°C for 1 hour. Centrifuge at 800g for 20 minutes. Transfer the supernatant to a new 1.5ml EP tube. Protect from light and store at -20°C until use.

[0048] Specific antibody titer determination: The specific IgG titer in the immune serum was determined by ELISA method, that is, the recombinant antigen protein to be detected was coated on a 96-well enzyme-labeled plate at 1 μg / ml, blocked with milk, and then a series of gradient dilutions of mouse immune serum were added. Horseradish peroxidase-labeled goat anti-mouse IgG enzyme-labeled antibody was used as the detection antibody. After color development, the OD was read on a microplate reader (Molecule Device). 450mm -OD 570mm Light absorption value.

[0049] The OD value of 0.105 was used as the cut-off value, that is, when the OD value of the serum group at a certain dilution was ≥ 0.105, it was positive; when the OD value was < 0.105, the previous dilution was the antibody titer of the serum.

[0050] Immunogenicity study results:

[0051] Table 1 Antibody titer test results after immunization of mice with single-component antigens of recombinant NHBA proteins with different modifications

[0052] immune antigens Antigen dose NHBA ELISA antibody titer NHBA 10 μg <![CDATA[3.20*10 4 ]]> P4-NHBA 10 μg <![CDATA[1.74*10 5 ]]>

[0053] The results showed that both recombinant NHBA proteins, when used alone to immunize mice, induced high levels of specific antibody responses, demonstrating good immunogenicity. The P4-NHBA antigen, modified with lipidation at the N-terminus by the addition of a P4 signal peptide, induced higher titers of specific antibodies in mice compared to the unmodified NHBA antigen, demonstrating improved immunogenicity.

Claims

1. P4-NHBA recombinant protein, which has the following structural characteristics from N-terminus to C-terminus: P4-linker-NHBA, that is, the N-terminus is the P4 lipoprotein signal peptide from non-typeable Haemophilus influenzae, the C-terminus is the NHBA recombinant protein from group B meningococcus, and the linker between the two represents the connecting peptide.

2. The P4-NHBA recombinant protein according to claim 1, characterized in that: (1) The sequence of P4 is shown in SEQ ID NO: 3; (2) Linker represents a connecting peptide, which has a length of 0 to 15 amino acid residues. When the length is 0, it means that no connecting peptide is required, that is, the front and back elements are directly covalently connected in series; (3) NHBA is obtained by removing the 1st to xth amino acid residues from the N-terminus of SEQ ID NO: 1 or a sequence having 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater identity thereto; When the linker length is 0, x=17; when the linker length is not 0, the N-terminus of the linker is cysteine, and 18≤x≤24.

3. The P4-NHBA recombinant protein according to claim 2, characterized in that The following amino acids or fragments are repeated downstream of the N-terminal cysteine of the linker peptide: G, GGGGS or EAAAK.

4. The P4-NHBA recombinant protein according to claim 2, characterized in that The amino acid sequence of the P4-NHBA recombinant protein is shown in SEQ ID NO:

4.

5. An immunogenic composition comprising the P4-NHBA recombinant protein according to any one of claims 1 to 4.

6. The immunogenic composition according to claim 5, characterized in that The amino acid sequence of the P4-NHBA recombinant protein is shown in SEQ ID NO:

4.

7. The immunogenic composition according to any one of claims 5 to 6, characterized in that The immunogenic composition further comprises an adjuvant, preferably an aluminum adjuvant. A polynucleotide encoding the P4-NHBA recombinant protein according to any one of claims 1 to 4.

9. The polynucleotide according to claim 7, characterized in that The polynucleotide comprises the sequence shown in SEQ ID NO:

6.

10. A recombinant expression vector comprising the polynucleotide according to any one of claims 8 to 9. A recombinant expression cell comprising the recombinant expression vector according to claim 10 .

12. Use of any one of the following (1) to (5) or a combination of any two or more thereof in the preparation of a medicament for preventing or alleviating Neisseria meningitidis infection and / or diseases caused by Neisseria meningitidis: (1) The P4-NHBA recombinant protein according to any one of claims 1 to 4; (2) The immunogenic composition according to any one of claims 5 to 7; (3) The polynucleotide according to any one of claims 8 to 9; (4) the recombinant expression vector according to claim 10; (5) The recombinant expression cell according to claim 11; Preferably, the Neisseria meningitidis is serogroup B Neisseria meningitidis.

13. The method for preparing the P4-NHBA recombinant protein according to any one of claims 1 to 4, characterized in that: The polynucleotide encoding the recombinant protein is cloned into an expression vector to obtain a recombinant expression vector, which is then transferred into a recipient cell to obtain a recombinant expression cell for fermentation culture, and separation and purification are performed to obtain the recombinant protein.

14. The preparation method according to claim 13, characterized in that The polynucleotide encoding the recombinant protein comprises the sequence shown in SEQ ID NO:

6.

15. The preparation method according to any one of claims 13 to 14, characterized in that: The recipient cell is Escherichia coli.

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