Group B meningococcus fusion protein based on iron nanoparticles as well as preparation method and application of group B meningococcus fusion protein

Abstract

The invention discloses a group B meningococcus fusion protein based on iron nanoparticles as well as a preparation method and application of the group B meningococcus fusion protein. The fusion protein comprises a ferritin subunit or a variant thereof, and an operably linked factor H binding protein (fHbp) or a variant thereof. The fusion protein has good immunogenicity, the immune response level is obviously increased compared with that of an fHbp monomer, and immune response aiming at heterologous or homologous group B meningococcus strains can be initiated. Furthermore, the fusion protein can be used as a candidate antigen of group B meningococcus, and has a very high reference value in the development of group B meningococcus vaccines.

Description

Technical Field

[0001] This application relates to the field of biomedical technologies, and specifically relates to an iron nanoparticle-based Neisseria meningitidis serogroup B fusion protein, its preparation method and application, and further relates to a fusion protein containing ferritin and factor H binding protein, a nucleic acid molecule encoding the same, a recombinant expression vector containing the nucleic acid molecule, a cell expressing the same, a vaccine preparation containing the same, its preparation method and application, etc. Background Art

[0002] Gram-negative bacterium Neisseria meningitidis can cause severe sepsis and meningococcal meningitis, and invasive meningococcal disease (IMD) is associated with a 5-15% mortality rate. Meningococcal serogroups are distinguished by the composition of their capsular polysaccharides. The five serogroups most commonly associated with invasive disease are A, B, C, W, and Y. Since the early 1990s, effective monovalent or multivalent polysaccharide conjugate vaccines against meningococcal serogroups A, C, W, and Y have emerged. However, due to the poor immunogenicity of the capsular polysaccharide of serogroup B (NmB) and the variability of the major outer membrane proteins, it has been a challenge to develop an effective serogroup B meningococcal vaccine.

[0003] Currently, two serogroup B meningococcal vaccines (Bexsero™ and Trumenba®) have been marketed, and the recombinant antigens in both vaccines have good immunogenicity. However, the outer membrane vesicles (OMVs) in the Bexsero™ vaccine and the lipoprotein components in Trumenba® are prone to causing side reactions to the body, and the human tolerance is poor. Therefore, to improve the immunogenicity and safety of recombinant antigens, a multimer is considered as a carrier protein, and the antigen is linked to the carrier protein to increase the immunogenicity by increasing the number of antigens and improve the safety by reducing the antigen dosage and reducing the occurrence of side reactions. There are many types of multimer carrier proteins, and ferritin is a multimer that has received extensive attention. Reports have shown the display of influenza virus antigen HA on the surface of ferritin nanoparticles, and subsequently reported the display of antigens on the surface of ferritin nanoparticles, the encapsulation of antigens in the nanoparticle cavity, or targeted drugs. Ferritin has been recognized as a highly potential carrier protein and a vehicle for delivering macromolecules.

[0004] Ferritin is present in almost all organisms, including bacteria, fungi, plants, and animals. It can self-assemble into nanoparticles and has been used as a delivery tool for a new type of recombinant epitope vaccine. Ferritin nanoparticles are composed of 24 subunits, forming a spherical structure with an outer diameter of 12 nm and an inner diameter of 8 nm, presenting 3-fold and 4-fold axes. Each subunit includes a four-α helix bundle (A-D) and a short E-α helix (located at the C-terminus). The ferritin complex also exhibits significant thermal and pH stability (tolerating temperatures up to 80 - 100 °C and a pH range of 3 - 10), monodispersity, small and uniform size, biocompatibility, biodegradability, low-cost large-scale production, and a cavity with reversible assembly / dissembly function. Summary of the Invention

[0005] Based on this, it is necessary to provide a group B meningococcal fusion protein based on ferritin nanoparticles, its preparation method, and its application.

[0006] The first aspect of the present application provides a fusion protein, which comprises a ferritin subunit or its variant, and an operably linked factor H-binding protein (fHbp) or its variant.

[0007] In some embodiments, the fusion mode of the fusion protein includes the connection of the factor H-binding protein or its variant to the N-terminus, C-terminus, or loop truncation of the ferritin subunit or its variant; optionally, the factor H-binding protein or its variant is connected to the C-terminus of the loop truncation of the ferritin subunit or its variant.

[0008] In some embodiments, the ferritin subunit variant includes truncating the ferritin subunit; optionally, truncating 1 - 4 amino acids from the N-terminus or 1 - 20 amino acids from the C-terminus of the ferritin subunit.

[0009] In some embodiments, the ferritin is selected from ferritin of mammals, amphibians, plants, or bacteria; optionally, the ferritin is selected from ferritin of bacteria; further optionally, the ferritin is Helicobacter pylori ferritin.

[0010] In some embodiments, the factor H-binding protein variant includes truncating the factor H-binding protein; optionally, truncating 1 - 26 amino acids from the N-terminus of the factor H-binding protein.

[0011] In some embodiments, the fusion protein comprises the ferritin subunit variant and the factor H-binding protein variant;

[0012] Optionally, the ferritin subunit variant and the factor H-binding protein variant are connected by a linker;

[0013] Further optionally, the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0014] The second aspect of the present application provides a nucleic acid molecule encoding the fusion protein described in the first aspect of the present application.

[0015] The third aspect of the present application provides a recombinant expression vector comprising the nucleic acid molecule described in the second aspect of the present application.

[0016] The fourth aspect of the present application provides a cell comprising the recombinant expression vector described in the third aspect of the present application.

[0017] The fifth aspect of the present application provides a method for preparing the fusion protein described in the first aspect of the present application, which comprises culturing the cell described in the fourth aspect of the present application; and obtaining the fusion protein from the culture.

[0018] The sixth aspect of the present application provides a vaccine preparation comprising the fusion protein described in the first aspect of the present application.

[0019] In some embodiments, the fusion protein comprises one or more of the fusion protein with the amino acid sequence as shown in SEQ ID NO: 3 and the fusion protein with the amino acid sequence as shown in SEQ ID NO: 4.

[0020] In some embodiments, the vaccine preparation further comprises an adjuvant, and the adjuvant comprises one or more of aluminum adjuvant, Freund's adjuvant, monophosphoryl lipid A, oil-in-water emulsion, AS04 adjuvant, and CpG oligonucleotide adjuvant;

[0021] The seventh aspect of the present application provides the use of the fusion protein described in the first aspect of the present application, the nucleic acid molecule described in the second aspect of the present application, the recombinant expression vector described in the third aspect of the present application, the cell described in the fourth aspect of the present application, or the vaccine preparation described in the sixth aspect of the present application in the preparation of a medicament for preventing or treating meningitis.

[0022] The fusion protein provided above has good immunogenicity, and the immune response level is significantly higher than that of the fHbp monomer, and the fusion protein can elicit an immune response against heterologous or homologous group B meningococcal strains. Further, this fusion protein can be used as a candidate antigen for group B meningococcus and has high reference value in the development of group B meningococcal vaccines. Description of the Drawings

[0023] To more clearly illustrate the technical solutions in the embodiments and examples of the present application and to more comprehensively understand the present application and its beneficial effects, the following will briefly introduce the accompanying drawings required for the description of the embodiments or examples. Obviously, the accompanying drawings in the following description are only some embodiments of the present application. For those skilled in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0024] Figure 1 SDS-PAGE identification of the fusion protein in an embodiment of the present application;

[0025] Figure 2 DLS identification of the fusion protein in an embodiment of the present application, where "size" represents the particle size, "intensity" represents the scattered light intensity, "Z-Average" represents the average particle size, "Pdl" represents the probability of light intensity fluctuation, "Intercept" represents the intercept, "result quality" represents the result quality, and "peak" represents the peak;

[0026] Figure 3 TEM identification of the fusion protein in an embodiment of the present application;

[0027] Figure 4 In A, it is the detection of the titer of the specific antibody induced by the FV1 fusion protein in an embodiment of the present application, Figure 4 In B, it is the detection of the titer of the specific antibody induced by the FV2 fusion protein in an embodiment of the present application, Figure 4 In C, it is the detection of the titer of the anti-fHbp antibody. Specific embodiments

[0028] To facilitate the understanding of the present application, the following will describe the present application more comprehensively with reference to the relevant accompanying drawings. The preferred embodiments of the present application are shown in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure content of the present application more thorough and comprehensive.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present application belongs. The terms used in the description of the present application in this specification are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0030] In this application, "optionally", "optional", and "option" mean having or not having, that is, any one selected from two alternative options of "having" or "not having". If "optional" appears multiple times in a technical solution, without special instructions and without contradictions or mutual restrictions, each "optional" is independent of each other.

[0031] In this application, "preferably", "better", "more preferably", and "should preferably" are only used to describe embodiments or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of this application.

[0032] The terms "having", "containing", "including", and "comprising" used in this application are synonyms, which are inclusive or open-ended and do not exclude additional, unmentioned members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features also include actions, conditions under which actions occur, timing, states, etc.

[0033] In this application, in a technical feature or technical solution described in an open language, it includes a closed technical feature or technical solution composed of the listed content, and also includes an open technical feature or technical solution containing the listed content.

[0034] In this application, for units related to data ranges, if only the unit is attached after the right endpoint, it means that the units of the left endpoint and the right endpoint are the same.

[0035] In this application, for a method process involving multiple steps, unless there are clear different descriptions in this article, the execution of these steps has no strict order limit, and it can be executed in other orders than the described one. Moreover, any one step can include multiple sub-steps or multiple stages. These sub-steps or stages do not necessarily need to be executed at the same moment, but can be executed at different moments, and their execution order does not necessarily need to be sequential, but can be executed alternately or simultaneously with other steps or a part of the sub-steps or stages of other steps.

[0036] In this application, for exemplary descriptions such as "in some embodiments (or examples)" and "in one embodiment (or example)", it can cover but is not limited to the following meanings: These solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0037] In this application, in "the first aspect", "the second aspect", "the third aspect", etc., the terms "first", "second", "third", etc. are only for descriptive purposes and cannot be construed as indicating or implying relative importance or quantity, nor can they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", etc. only serve the purpose of non-exhaustive enumerative description and should be understood not to constitute a closed limitation on quantity.

[0038] In this application, regarding numerical intervals (i.e., numerical ranges), unless otherwise specified, the distribution of the selectable numerical values within the numerical interval is considered continuous and includes the two numerical endpoints of the numerical interval (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. The "numerical interval" allows for a broad inclusion of numerical interval types such as percentage intervals, ratio intervals, ratio value intervals, etc.

[0039] Currently, due to the poor immunogenicity of the capsular polysaccharide of serogroup B (NmB) and the variability of the major outer membrane proteins, the development of an effective meningococcal serogroup B vaccine has always been a challenge. For the two currently marketed meningococcal serogroup B vaccines (Bexsero™ and Trumenba®), although the recombinant antigens in both vaccines have good immunogenicity, the outer membrane vesicles (OMVs) in the Bexsero™ vaccine and the lipoprotein components in the Trumenba® are prone to cause side reactions in the body and the human tolerance is poor. Therefore, to improve the immunogenicity and safety of the recombinant antigen, a multimer is considered as a carrier protein, the antigen is linked to the carrier protein, the immunogenicity is increased by increasing the amount of antigen, and the safety is improved by reducing the amount of antigen used, and the occurrence of side reactions is reduced. There are many types of multimer carrier proteins, among which ferritin is a multimer that has received extensive attention. Linking fHbp to iron nanoparticles can not only improve the immunogenicity, but also reduce the amount of antigen used, improve the safety, and reduce the occurrence of side reactions. Based on this, the embodiments of this application at least provide a fusion protein containing ferritin subunits and factor H binding protein, and its preparation method and application.

[0040] In one embodiment of the present application, in view of the relatively weak immunogenicity exhibited by the monomer of the Neisseria meningitidis serogroup B candidate antigen fHbp, taking advantage of the characteristic that ferritin subunits can self-assemble into nanoparticles, the antigen is displayed on the surface of iron nanoparticles, thereby enhancing the immunogenicity of the antigen.

[0041] In a first aspect of the present application, there is provided a fusion protein comprising a ferritin subunit or a variant thereof, and a factor H binding protein (fHbp) or a variant thereof operably linked.

[0042] In the present application, unless otherwise specified, the term "variant" refers to a protein obtained by mutating the wild type by one or any combination of means including but not limited to amino acid substitution, deletion (including truncation), insertion, translocation, permutation, and modification. Taking the variant of fHbp as an example, it comprises some or all of the functions possessed by the wild-type fHbp, or it at least comprises the functions possessed by the wild-type fHbp.

[0043] In the present application, unless otherwise specified, the term "operably linked" refers to the functional relationship between two regions of the fusion protein, namely the ferritin subunit or a variant thereof, and fHbp or a variant thereof; wherein the two regions are linked to produce the fusion protein.

[0044] In some embodiments, the fusion mode of the fusion protein includes the linking of the factor H binding protein or a variant thereof with the N-terminus, C-terminus, or loop truncation body of the ferritin subunit or a variant thereof.

[0045] In some embodiments, the factor H binding protein or a variant thereof is linked to the C-terminus of the loop truncation body of the ferritin subunit or a variant thereof.

[0046] In some embodiments, the ferritin subunit variant includes truncating the ferritin subunit.

[0047] In some embodiments, the ferritin subunit is truncated by 1 to 4 amino acids from the N-terminus or 1 to 20 amino acids from the C-terminus. Further, the ferritin subunit is truncated by 4 amino acids from the N-terminus or 20 amino acids from the C-terminus.

[0048] In some embodiments, the ferritin is selected from ferritins of mammals, amphibians, plants, or bacteria; further, the ferritin is selected from ferritins of bacteria; still further, the ferritin is Helicobacter pylori ferritin.

[0049] In some embodiments, the factor H binding protein variant includes truncating the factor H binding protein.

[0050] In some embodiments, the factor H binding protein is truncated by 1 to 26 amino acids from the N-terminus. Further, the factor H binding protein is truncated by 26 amino acids from the N-terminus.

[0051] In some embodiments, the fusion protein comprises a ferritin subunit variant and an H factor binding protein variant.

[0052] In some embodiments, the ferritin subunit variant and the H factor binding protein variant are linked by a linker.

[0053] In some embodiments, the amino acid sequence of the linker can be (GGGGS)n, (GGGS)n, (GGS)n, (GS)n, (AS)n, (G)n or (A)n, where n is selected from 1, 2, 3, 4, 5 or 6. Further, the amino acid sequence of the linker is GGGGS.

[0054] In some embodiments, the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 3 or the amino acid sequence has at least 80% (such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identity to SEQ ID NO: 3.

[0055] In some embodiments, the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 4 or the amino acid sequence has at least 80% (such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identity to SEQ ID NO: 4.

[0056] In this application, unless otherwise specified, "identity" refers to the percentage of amino acid residues in a first sequence that are identical to the amino acid residues in a second sequence when aligning the amino acid sequences (introducing gaps if necessary) to achieve the maximum percentage of sequence identity and not considering any conservative substitutions as part of the sequence identity. To determine the percentage of amino acid sequence identity, the alignment can be achieved in a variety of ways within the scope of those skilled in the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring the alignment, including any algorithms required to achieve the maximum alignment over the entire length of the sequences being compared.

[0057] In the second aspect of this application, there is provided a nucleic acid molecule encoding the fusion protein of the first aspect of this application.

[0058] In the present application, unless otherwise specified, "nucleic acid molecule" mainly refers to an isolated nucleic acid molecule. "Isolated" means that the molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth medium. Generally, the term "isolated" does not intend to mean the complete absence of these materials or the absence of water, buffer, or salt, unless they are present in an amount that significantly interferes with the experimental or therapeutic use of the compounds described herein.

[0059] In the third aspect of the present application, there is provided a recombinant expression vector comprising the nucleic acid molecule of the second aspect of the present application.

[0060] In some embodiments, the recombinant expression vector can be obtained by conventional methods in the art. For example, it can be constructed by ligating the nucleic acid molecule described in the present application to various expression vectors. The expression vector is capable of accommodating the nucleic acid molecule described in the present application. For example, the expression vector can include a plasmid, cosmid, phage, or viral vector.

[0061] In the fourth aspect of the present application, there is provided a cell comprising the recombinant expression vector of the third aspect of the present application.

[0062] In the present application, unless otherwise specified, "cell", also namely "host cell", refers to a cell into which an expression vector has been introduced. Host cells can include bacteria, microorganisms, plant, or animal cells. Bacteria that are easily transformable include members of the Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0063] In the fifth aspect of the present application, there is provided a method for preparing the fusion protein of the first aspect of the present application, which includes culturing the cell of the fourth aspect of the present application; and obtaining the fusion protein from the culture.

[0064] In the sixth aspect of the present application, there is provided a vaccine preparation comprising the fusion protein of the first aspect of the present application.

[0065] In some embodiments, the fusion protein comprises one or more of a fusion protein having an amino acid sequence such as that set forth in SEQ ID NO: 3 and a fusion protein having an amino acid sequence such as that set forth in SEQ ID NO: 4.

[0066] In some embodiments, the vaccine formulation further comprises an adjuvant, and the adjuvant comprises one or more of an aluminum adjuvant, Freund's adjuvant, monophosphoryl lipid A, oil-in-water emulsion, AS04 adjuvant, and CpG oligonucleotide adjuvant. Further, the adjuvant is selected from aluminum adjuvants, and still further, the aluminum adjuvant is aluminum hydroxide.

[0067] In the present application, unless otherwise specified, an "adjuvant" refers to a substance that non-specifically enhances or boosts the immune response to an immunogenic agent in an individual subject exposed to a mixture when added to the immunogenic agent such as an antigen.

[0068] The seventh aspect of the present application provides the use of the fusion protein of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the recombinant expression vector of the third aspect of the present application, the cell of the fourth aspect of the present application, or the vaccine formulation of the sixth aspect of the present application in the preparation of a medicament for preventing or treating meningitis.

[0069] Some examples are provided below.

[0070] The embodiments of the present application will be described in detail below in conjunction with the examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specified conditions in the following examples, priority is given to the guidance given in the present application, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturers, or referring to the experimental methods known in the art.

[0071] In the following examples, for the measurement parameters of the raw material components, unless otherwise specified, there may be slight deviations within the weighing accuracy range. For the temperature and time parameters, acceptable deviations caused by the instrument test accuracy or operation accuracy are allowed.

[0072] The amino acid sequences involved in the following examples are shown below:

[0073] SEQ ID NO: 1

[0074] VAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGKLITLESGEFQVYKQSHSALTALQTEQVQDSEDSGKMVAKRQFRIGDIAGEHTSFDKLPKGGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVELATAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGQAQEVAGSAEVETANGIHHIGLAAKQ

[0075] SEQ ID NO: 2

[0076] VAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ

[0077] SEQ ID NO: 3

[0078] MLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIVFLNENNVPVQLTSISAPEHKFESLTQIFQKAYEHEQHISESINNIVDHAIKGKDHATFNFLQWYVSEQHEEEVLFKDILDKIELIGNEGGGGSVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGKLITLESGEFQVYKQSHSALTALQTEQVQDSEDSGKMVAKRQFRIGDIAGEHTSFDKLPKGGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVELATAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGQAQEVAGSAEVETANGIHHIGLAAKQ

[0079] SEQ ID NO: 4

[0080] MLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIVFLNENNVPVQLTSISAPEHKFESLTQIFQKAYEHEQHISESINNIVDHAIKGKDHATFNFLQWYVSEQHEEEVLFKDILDKIELIGNEGGGGSVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ

[0081] Example 1

[0082] 1. Design and Preparation of fHbp-Ferritin Subunit Fusion Protein

[0083] 1) Select the fHbp amino acid sequences of two variants, V1.13 and V2.16, from the PubMLST database, and simultaneously delete the leader peptide AA1-AA26 at the N-terminus of fHbp. The specific sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0084] 2) Connect fHbp to the C-terminus of the loop-truncated ferritin subunit (abbreviated as FV1 and FV2) by gene fusion, insert a GGGGS linker between the two proteins, and then clone the fusion fragment into the expression vector pET24b with a 6×his tag at the N-terminus. Obtain the recombinant plasmid of this fusion fragment through gene synthesis commissioned by General Biosystems. The specific amino acid sequences of FV1 and FV2 are shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively.

[0085] 3) Transform the recombinant plasmid into the Escherichia coli expression strain BL21(DE3), pick monoclonal colonies into a shaking tube containing LB medium with the corresponding antibiotic, and shake at 37°C and 200 rpm overnight. The next day, transfer it to fresh LB medium with the corresponding antibiotic, shake at 37°C and 200 rpm until the OD600 of the bacterial solution reaches 0.6 - 0.8, add 1 mM IPTG (isopropyl-β-D-thiogalactoside), and continue to culture at 37°C and 180 rpm for 4 h. Centrifuge to collect the bacterial cells and perform SDS-PAGE identification.

[0086] 4) After purification by three steps of nickel column affinity chromatography, anion exchange chromatography, and gel filtration, identify the fHbp fusion protein with a purity of over 90% through SDS-PAGE and HPLC. The results are as Figure 1 shown. The concentrations of the fusion proteins FV1 and FV2 measured by the Lowary method are 0.89 mg / mL and 0.73 mg / mL respectively.

[0087] 2. Identification of the size and morphology of the fHbp-iron fusion protein

[0088] 1) Use DLS (dynamic light scattering) to detect the particle size of the purified fHbp-iron fusion protein. The results are as Figure 2 shown.

[0089] 2) Dilute the fHbp-iron fusion protein with PBS buffer to a concentration of 0.5 mg / mL and send it to a CRO company for TEM (transmission electron microscopy) identification. The results are as Figure 3 shown. It can be seen from the DLS graph and TEM graph that the fusion protein formed by fHbp and the ferritin subunit forms nanoparticles with appropriate particle size (about 26 nm) and morphology (relatively uniform spherical shape).

[0090] 3. Vaccine formulation

[0091] 1) Prepare the formulation according to the human dose. Adsorb the fHbp iron fusion protein stored in physiological saline onto aluminum hydroxide adjuvant, where the fHbp iron fusion protein is 10 μg, aluminum hydroxide is 0.5 mg, and the buffer system is histidine buffer (pH 6.0).

[0092] 2) For the combined vaccine of two fHbp iron fusion proteins FV1 and FV2, the masses of the two proteins are 10 μg respectively, and the other vaccine components are the same as above.

[0093] 3) Identify the particle size and detect the endotoxin content of the prepared preparation.

[0094] 4. Animal immunization

[0095] 1) Select female mice of CD1 strain at 4 - 6 weeks old, 5 mice per group, and perform intraperitoneal injection at 1 / 5 of the human dose.

[0096] 2) Immunize on days 0 and 28 respectively, and collect blood on days 28 and 56 respectively to collect serum.

[0097] 5. Antibody level detection

[0098] 1) Detect the specific antibody titer of serum by conventional ELISA. That is, first coat a 96 - well plate with purified fusion protein, after blocking, sequentially add primary antibody sera with different dilution multiples for incubation, enzyme - labeled secondary antibody incubation, add enzyme detection substrate, and perform result interpretation and data analysis.

[0099] 2) Detect the bactericidal antibody titer of serum by SBA: First spread Neisseria meningitidis serogroup B strains on blood agar plates, culture overnight at 37 °C and 5% CO2, inoculate single colonies into Mueller - Hinton medium to control the initial OD620 of the bacterial solution at 0.05 - 0.08, culture in a shaker at 37 °C until OD620 reaches 0.23 - 0.24, and measure the bacterial viability. All mouse sera to be tested are first heat - inactivated at 56 °C for 30 min. The total volume in each well is 50 μL, including 25 μL of serially two - fold diluted test serum, 12.5 μL of bacterial working solution, and 12.5 μL of human complement. Controls include: serum incubated with complement serum, immune serum incubated with bacteria, and inactivated complement. Immediately after adding complement, take 10 μL of the control and spread it on a Mueller - Hinton agar plate, incubate at 37 °C and 5% CO2 for 1 h. Take 7 μL of each sample and spot it on a Mueller - Hinton agar plate, incubate at 37 °C and 5% CO2 for 18 h.

[0100] The detection results of specific antibody titers are shown in Figure 4 , from Figure 4 A and Figure 4As can be seen from B, whether the prepared fHbp fusion protein is immunized alone or in combination, the titers of specific antibodies induced in the body are significantly higher than those immunized with fHbp monomer, and the titers of the former all reach nearly 1:10 7 , showing good immunogenicity. In addition, from Figure 4 As can be seen from C, the titer of anti-fHbp antibody in the immune serum of fHbp fusion protein is significantly higher than that of anti-ferritin antibody, indicating that ferritin has no inhibitory effect on the immunogenicity of this fusion protein.

[0101] The results of the detection of bactericidal antibody titers are shown in Table 1. It can be seen that the fHbp fusion protein can induce functional antibodies with obvious bactericidal activity in the body (titer above 1:4). Especially, after the combined immunization of the two fHbp fusion proteins, a broader and higher protective response can be induced, and the bactericidal coverage rate reaches about 80%.

[0102] Table 1

[0103]

[0104] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0105] The above-described embodiments only represent several implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several deformations and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims, and the specification and drawings can be used to explain the scope of the claims.

Claims

1. A fusion protein, characterized in that It comprises a ferritin subunit or a variant thereof, and an operably linked factor H binding protein (fHbp) or a variant thereof.

2. The fusion protein according to claim 1, characterized in that The fusion method of the fusion protein includes connecting the factor H binding protein or its variant to the N-terminus, C-terminus or loop truncation of the ferritin subunit or its variant; Optionally, the factor H binding protein or its variant is linked to the C-terminus of the loop truncation of the ferritin subunit or its variant.

3. The fusion protein according to claim 2, characterized in that The ferritin subunit variant comprises truncating the ferritin subunit; optionally, the ferritin subunit is truncated by 1 to 4 amino acids from the N-terminus or 1 to 20 amino acids from the C-terminus.

4. The fusion protein according to claim 3, characterized in that The ferritin is selected from ferritin of mammals, amphibians, plants or bacteria; optionally, the ferritin is selected from ferritin of bacteria; further optionally, the ferritin is Helicobacter pylori ferritin.

5. The fusion protein according to any one of claims 1 to 4, characterized in that The factor H binding protein variant comprises truncating the factor H binding protein; optionally, the N-terminus of the factor H binding protein is truncated by 1 to 26 amino acids.

6. The fusion protein according to claim 5, characterized in that It comprises the ferritin subunit variant and the factor H binding protein variant; Optionally, the ferritin subunit variant and the factor H binding protein variant are connected via a linker; Further optionally, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 3 or SEQ ID NO:

4.

7. A nucleic acid molecule, characterized in that It encodes the fusion protein according to any one of claims 1 to 6.

8. A recombinant expression vector, characterized in that: It comprises the nucleic acid molecule as claimed in claim 7.

9. A cell, characterized in that It comprises the recombinant expression vector according to claim 8.

10. A method for preparing the fusion protein according to any one of claims 1 to 6, characterized in that: The method comprises culturing the cell according to claim 9; and obtaining the fusion protein from the culture.

11. A vaccine preparation, characterized in that It comprises the fusion protein according to any one of claims 1 to 6; Optionally, the fusion protein includes one or more of a fusion protein having an amino acid sequence as shown in SEQ ID NO: 3 and a fusion protein having an amino acid sequence as shown in SEQ ID NO: 4; Further optionally, the vaccine preparation also includes an adjuvant, and the adjuvant includes one or more of aluminum adjuvant, Freund's adjuvant, monophosphoryl lipid A, oil-in-water emulsion, AS04 adjuvant and CpG oligonucleotide adjuvant.

12. Use of the fusion protein according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the recombinant expression vector according to claim 8, the cell according to claim 9 or the vaccine preparation according to claim 11 in the preparation of a medicament for preventing or treating meningitis.

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