Specific binding protein of fHbp as well as production method and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to fHbp specific binding protein and a production method and application thereof. The specific binding protein has an antigen binding structural domain, and the antigen binding structural domain has HCDR1 to HCDR3 as shown in SEQ ID NO.5 to 7, and LCDR1 to LCDR3 as shown in SEQ ID NO.8 to 10. The specific binding protein can be used for preparing a specific binding protein. The fHbp specific binding protein provided by the invention has pertinence to group B meningococcus fHbp-V2, does not have cross recognition reaction with a subgroup B, does not recognize escherichia coli host protein, can be used for antigen qualitative and quantitative detection, can be used for identification, qualitative and quantitative detection of fHbp-V2 and V3 antigens in a multi-component group B meningococcus vaccine containing fHbp-V2 and V3 antigens, and has the advantages of high specificity, high sensitivity, high sensitivity and the like. And the method can also be used for identifying the expression abundance of meningococcus strains V2 and V3.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to a specific binding protein of fHbp and a production method and application thereof. Background Art

[0002] Neisseria meningitidis group B, commonly known as serogroup B meningococcus, can cause epidemic meningitis and sepsis, which are associated with high morbidity and mortality. As a common serogroup of invasive meningitis, serogroup B has become the predominant strain, accounting for over 90% of cases along with serogroups A and C.

[0003] Unlike other meningococci, the capsular polysaccharide component of serogroup B meningococci is a homologous polymer of α(2-8)N-acetylneuraminic acid (polysialic acid) and an autoantigen, similar to α2-8 sialylated human glycoproteins, such as adhesion molecules found in fetal neural cells. Similarly, the lipopolysaccharide structure of many meningococci contains a terminal N-lactose moiety similar to liposugar found in human cells, posing a risk of inducing autoimmune disease. Therefore, outer membrane proteins of serogroup B Neisseria meningitidis have become candidate vaccine antigens. Its outer membrane lipoprotein 2086, or LP2086, is known as factor H binding protein (fHbp) because it binds to human factor H, downregulating the alternative complement activation pathway and allowing the bacterium to evade recognition and attack.

[0004] fHbp is a specific lipoprotein expressed on the surface of nearly all Neisseria meningitidis bacteria, with a relative molecular mass of approximately 29 kDa. fHbp is divided into three variants: variant 1 (V1), variant 2 (V2), and variant 3 (V3). V1 is also known as subgroup B, while V2 and V3 belong to subgroup A. Currently, V1 and V2 strains predominate among group B meningococci prevalent both domestically and internationally. fHbp amino acid sequence alignments show that the fHbp sequence is highly conserved, with different subvariants within the same subfamily sharing 91.6% to 100% identity and approximately 62.8% identity across different subfamilies.

[0005] fHbp is an important virulence factor and vaccine antigen of Neisseria meningitidis. It is an important component of group B meningococcal vaccines that are already on the market or under development. Therefore, how to achieve its detection is crucial to vaccine quality control. Summary of the Invention

[0006] Based on this, one or more embodiments of the present application provide a specific binding protein for fHbp, a production method, and an application thereof, including the following technical solutions:

[0007] One or more embodiments of the present application provide a specific binding protein for fHbp, wherein the specific binding protein has an antigen binding domain, wherein the antigen binding domain has HCDR1 to HCDR3 shown in SEQ ID NOs. 5 to 7, and LCDR1 to LCDR3 shown in SEQ ID NOs. 8 to 10.

[0008] In some embodiments of the present application, the specific binding protein is an antibody, an antigen-binding fragment of an antibody, or a small modular immune drug;

[0009] Optionally, the specific binding protein is a monoclonal antibody, a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a Fv fragment, a scFv fragment, a linear antibody, a multispecific antibody, a mini antibody, a chelated recombinant antibody, an internal antibody, a binding domain immunoglobulin fusion protein, a small modular immune drug, a camelized antibody or an antibody containing VHH.

[0010] In some embodiments of the present application, the species origin of the heavy chain framework region and the light chain framework region of the antigen binding domain are independently cattle, horses, pigs, sheep, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese or humans.

[0011] In some embodiments of the present application, the antigen-binding domain has a heavy chain variable region shown in SEQ ID NO.2 and a light chain variable region shown in SEQ ID NO.4.

[0012] In some embodiments of the present application, the specific binding protein further comprises a heavy chain constant region and / or a light chain constant region;

[0013] Optionally, the sequences of the heavy chain constant region and the light chain constant region are each independently selected from the sequence of any one of the constant regions of IgG, IgA, IgM, IgE and IgD.

[0014] In some embodiments of the present application, the species origin of the heavy chain constant region and the light chain constant region of the specific binding protein are independently cow, horse, pig, sheep, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose or human.

[0015] One or more embodiments of the present application provide the use of the fHbp-specific binding protein in preparing a detection kit.

[0016] One or more embodiments of the present application provide a detection kit, which includes the specific binding protein of fHbp.

[0017] In some embodiments of the present application, the detection kit further comprises other types of fHbp-specific binding proteins.

[0018] One or more embodiments of the present application provide a method for detecting fHbp or a strain expressing the same, wherein the method uses the specific binding protein or the detection kit to detect fHbp or a strain expressing the same.

[0019] In some embodiments of the present application, the fHbp belongs to subfamily A.

[0020] In some embodiments of the present application, the fHbp includes one or more of fHbp variant 2 and fHbp variant 3.

[0021] One or more embodiments of the present application provide a nucleic acid encoding the specific binding protein of fHbp.

[0022] In some embodiments of the present application, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown as SEQ ID NO.1, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown as SEQ ID NO.3.

[0023] One or more embodiments of the present application provide a vector comprising the nucleic acid.

[0024] In some embodiments of the present application, the vector is a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus or a mammalian cell virus.

[0025] One or more embodiments of the present application provide a cell, wherein:

[0026] Expressing the specific binding protein of fHbp; or,

[0027] Including the nucleic acid or the vector.

[0028] In some embodiments of the present application, the cell is a CHO cell, a COS cell, a NSO cell, a HeLa cell, a BHK cell or a HEK293 cell.

[0029] One or more embodiments of the present application provide a method for constructing the cell, which includes the step of introducing the nucleic acid or the vector into the cell to be transformed.

[0030] One or more embodiments of the present application provide a method for producing the fHbp-specific binding protein, wherein the method uses the cells to produce the fHbp-specific binding protein.

[0031] The details of one or more embodiments of the present application are set forth in the description below, and other features, objects, and advantages of the application will become apparent from the description and from the claims thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0033] Figure 1 For SDS-PAGE identification of fHbp-V2;

[0034] Figure 2 SDS-PAGE identification of monoclonal antibodies;

[0035] Figure 3 Standard curves established for antigen quantification assays;

[0036] Figure 4 To verify the specificity of the antigen quantitative detection method. DETAILED DESCRIPTION

[0037] Below in conjunction with accompanying drawing, embodiment and example, the application is described in further detail.It should be understood that these embodiment and example are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms and is not limited to the embodiment and example described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large amount of specific details are given in order to provide a more complete understanding of the application.It should be understood that the application can be implemented without one or more of these details.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing embodiments and examples only and are not intended to limit this application.

[0039] the term

[0040] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0041] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0042] In this application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0043] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0044] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0045] Herein, "preferred", "better", "more preferred" and "suitable" are merely used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of this application.

[0046] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0047] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0048] In this application, the terms "first," "second," "third," "fourth," etc. in "the first aspect," "the second aspect," "the third aspect," "the fourth aspect," etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should they be understood as implicitly indicating the importance or quantity of the indicated technical features. Furthermore, "first," "second," "third," "fourth," etc. serve only as non-exhaustive enumeration and description, and should be understood not to constitute a closed-ended limitation on quantity.

[0049] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0050] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0051] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0052] In this application, % (w / w) and wt% both refer to weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0053] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0054] In a first aspect of an embodiment of the present application, a specific binding protein of fHbp is provided, wherein the specific binding protein has an antigen binding domain, wherein the antigen binding domain has HCDR1 to HCDR3 shown in SEQ ID NOs. 5 to 7, and LCDR1 to LCDR3 shown in SEQ ID NOs. 8 to 10.

[0055] The fHbp-specific binding protein of the present application is specific for group B meningococcal fHbp-V2, has no cross-recognition reaction with the B subfamily, and does not recognize Escherichia coli host proteins. It can be used for qualitative and quantitative detection of antigens, and can be used for the identification, qualitative and quantitative detection of fHbp-V2 and V3 antigens in multi-component group B meningococcal vaccines containing fHbp-V2 and V3 antigens (accuracy controlled at 86%-110%). It can also be used to identify the expression abundance of V2 and V3 in meningococcal strains.

[0056] It is well known in the art that the binding specificity and affinity of an antibody are primarily determined by its CDR sequence. Based on mature, well-known existing technologies, the amino acid sequence of non-CDR regions can be easily altered to obtain variants with similar biological activity. Therefore, the present invention also encompasses "functional derivatives" of the binding protein. "Functional derivatives" refer to variants with amino acid substitutions, where a functional derivative retains detectable binding protein activity. "Functional derivatives" may include "variants" and "fragments," which have identical CDR sequences to the binding protein of the present invention and therefore possess similar biological activity.

[0057] The antigen-binding domains described herein may comprise one or more amino acid substitutions, deletions, or insertions relative to the CDR sequences described above, for example, the number of amino acid insertions, substitutions, or deletions may not exceed three, preferably one. Substitutions, deletions, or insertions may be introduced into nucleic acid molecules encoding the binding proteins of the present invention by conventional techniques such as site-directed mutagenesis or PCR-mediated mutagenesis. In some embodiments, conservative amino acid substitutions are made at one or more sites. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acids having similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0058] In some embodiments of the present application, the specific binding protein is an antibody, an antigen-binding fragment of an antibody, or a small modular immunopharmaceutical; alternatively, the specific binding protein is a monoclonal antibody, a F(ab')2 fragment, a Fab' fragment, a Fab fragment, an Fv fragment, a scFv fragment, a linear antibody, a multispecific antibody, a minibody, a chelated recombinant antibody, an intrabody, a binding domain immunoglobulin fusion protein, a small modular immunopharmaceutical, a camelized antibody, or an antibody containing VHH. In some embodiments, the antigen-binding fragment is derived from a complete antibody molecule, such as a monoclonal antibody. Preparation methods are known in the art.

[0059] The term "antibody" or "Ab" generally refers to a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. The light chains of an antibody can be classified as either kappa or lambda. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, which define the antibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively. In both light and heavy chains, the variable region is connected to the constant region by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (called complementary determining regions, CDRs) separated by relatively conserved regions (called framework regions, FRs). Each VH and VL consists of three CDRs and four FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from N-terminus to C-terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen binding site / part, respectively. The distribution of amino acids in various regions or domains follows the numbering definitions in common systems such as Kabat, IMGT, or Chothia. In the specific embodiments of the present disclosure, the CDR sequences are determined using the numbering definitions in the Kabat system.

[0060] The term "monoclonal antibody" or "monoclonal antibody" or "monoclonal antibody composition" refers to an antibody molecular product composed of a single molecule. The monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope. In one embodiment of the present application, the specific binding protein is a monoclonal antibody, which is a group B meningococcal fHbp-V2 specific monoclonal antibody that can only recognize subgroup A and has no cross-reaction with subgroup B (V1). It can be used in scenarios such as strain identification, antigen differentiation and quantitative detection.

[0061] The term "bispecific" or "bispecific" molecule refers to a molecule that specifically binds to two target molecules, or two different epitopes on the same target molecule. Bispecific molecules include bispecific antibodies that specifically bind to CD3 and a disease-associated antigen as described herein. In contrast, a "monospecific" molecule refers to a molecule that specifically binds to a single target molecule, particularly a single epitope on a single target molecule, such as the monoclonal antibody that binds to CD3 as described herein.

[0062] "Nanoantibodies" are generally defined as in WO 2008 / 020079 or WO 2009 / 138519, and in a specific aspect generally refer to VHH, humanized VHH or camelized VH (such as camelized human VH), or generally refer to sequence-optimized VHH (for example, optimized for chemical stability and / or solubility, maximum overlap with known human framework regions, and maximum expression). "Nanoantibodies" are obtained by genetic engineering methods and are mainly divided into three categories. The first category is the heavy chain variable region obtained from camelid HCAb, which is a single folding unit that retains complete antigen binding activity and is the smallest natural antibody fragment. The second category is the heavy chain variable region obtained from cartilaginous fish IgNARs such as sharks, represented by VNAR. The third category is the heavy chain or light chain variable region obtained from human or mouse monoclonal antibodies, which retains antigen binding activity, but has greatly reduced affinity and solubility. In one example, the specific binding protein for CD3 provided in the embodiments of the present application can be a nanoantibody. Nanobodies are the VHH domains of heavy-chain antibodies found in the serum of camelids and other species. With molecular weights as low as 12-15 kDa, only one-tenth the size of traditional antibodies, they are the smallest functional single-domain antibodies currently available that can stably bind to antigens. Due to their small molecular weight, strong tissue penetration, high specific affinity, ability to recognize gap epitopes, low immunogenicity, high water solubility, strong stability, simple production, ease of expression, and amenable engineering modifications, nanobodies can be used as affinity capture reagents, biosensors, and in vivo imaging tracers, and have broad application prospects in scientific research, substance detection, disease diagnosis, and treatment.

[0063] In some embodiments of the present application, the species origin of the heavy chain framework region and the light chain framework region of the antigen binding domain are independently cattle, horses, pigs, sheep, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese or humans.

[0064] In some embodiments of the present application, the antigen binding domain has a heavy chain variable region shown in SEQ ID NO.2 (or at least 80% identity with SEQ ID NO.2, for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and a light chain variable region shown in SEQ ID NO.4 (or at least 80% identity with SEQ ID NO.4, for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%).

[0065] Amino acid sequence "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 the amino acid sequences are aligned (introducing gaps if necessary) to achieve maximum sequence identity percentage, and not considering any conservative substitutions as part of the sequence identity. To determine amino acid sequence identity percentage, alignment can be achieved in a variety of ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine parameters suitable for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequences.

[0066] In some embodiments of the present application, the specific binding protein further comprises a heavy chain constant region and / or a light chain constant region;

[0067] Optionally, the sequences of the heavy chain constant region and the light chain constant region are each independently selected from the sequence of any one of the constant regions of IgG, IgA, IgM, IgE and IgD.

[0068] In some embodiments of the present application, the species origin of the heavy chain constant region and the light chain constant region of the specific binding protein are independently cow, horse, pig, sheep, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose or human.

[0069] As used herein, "skeleton," "framework," or "FR" regions refer to regions of an antibody variable domain excluding those defined as CDRs. Each antibody variable domain framework can be further subdivided into adjacent regions (FR1, FR2, FR3, and FR4) separated by CDRs. Typically, the variable regions VL and VH of the heavy and light chains are arranged in the following combinations of CDRs and FRs: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0070] The antibodies or fragments described herein may be humanized, where they comprise at least one human framework region; further optionally, the sequences of the heavy chain framework region and the light chain framework region are all antibody framework region sequences derived from humans. For example, one or more (e.g., one, two, three, four, five, or six) framework regions of an antibody produced by a hybridoma may be replaced with one or more (e.g., one, two, three, four, five, or six) human framework regions. Humanized antibodies are generally less immunogenic to humans than non-humanized antibodies, and therefore may offer therapeutic advantages in certain cases. Various human framework regions are known to those skilled in the art. Methods for preparing humanized antibodies are known in the art.

[0071] The antibodies or antigen-binding fragments of antibodies described herein may be chimeric, in which case they comprise at least one human constant region. For example, the constant region of an antibody produced by a hybridoma of the present invention may be replaced with a human constant region. Chimeric antibodies are generally less immunogenic in humans than non-chimeric antibodies and, therefore, may offer therapeutic advantages in certain situations. In some embodiments, the chimeric antibodies described herein comprise an IgG constant region. Various human constant regions are known to those skilled in the art. Methods for preparing chimeric antibodies are known in the art.

[0072] The second aspect of the embodiments of the present application provides the use of the fHbp-specific binding protein in preparing a detection kit.

[0073] According to a third aspect of the embodiments of the present application, a detection kit is provided, comprising the specific binding protein of fHbp.

[0074] In some embodiments of the present application, the detection kit further comprises other types of fHbp-specific binding proteins.

[0075] In a fourth aspect of the embodiments of the present application, a method for detecting fHbp or a strain expressing the same is provided. The method uses the specific binding protein or the detection kit to detect fHbp or a strain expressing the same.

[0076] In some embodiments of the present application, the fHbp belongs to subfamily A.

[0077] In some embodiments of the present application, the fHbp includes one or more of fHbp variant 2 and fHbp variant 3.

[0078] The detection kit and detection method of the present application can be based on the double antibody sandwich method to detect fHbp or its expressing strain. The double antibody sandwich method can be immunochromatography or chemiluminescence immunoassay (CLIA).

[0079] The detection kit and detection method of the present application can also be used to detect fHbp or the strain expressing it based on immunoturbidimetry.

[0080] In a fifth aspect of the embodiments of the present application, a nucleic acid is provided, wherein the nucleic acid encodes the specific binding protein of the fHbp.

[0081] The nucleic acids and nucleic acid molecules of the present application primarily refer to isolated nucleic acid molecules. "Isolated" refers to molecules that are substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth medium. Generally, the term "isolated" is not intended to refer to the complete absence of these materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compounds as described herein.

[0082] In some embodiments of the present application, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID NO.1 (or there is at least 80% identity with SEQ ID NO.1, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID NO.3 (or there is at least 80% identity with SEQ ID NO.3, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%).

[0083] In a sixth aspect of the embodiments of the present application, a vector is provided, wherein the vector comprises the nucleic acid.

[0084] The term "vector", which may also be referred to as "nucleic acid construct", refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which another DNA segment can be connected. Another type of vector is a viral vector, in which an additional DNA segment can be connected to a viral genome. Certain vectors can replicate autonomously in the host cell into which they are introduced (for example, bacterial vectors with bacterial replication origins and episomal mammalian vectors). Other vectors (for example, non-additive mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicate together with the host genome. In addition, certain vectors can direct the expression of the gene to which they are effectively connected. This vector is referred to as a "recombinant expression vector" (or simply "expression vector") in this article. Typically, expression vectors useful in recombinant DNA technology typically exist in the form of a plasmid. However, other forms of expression vectors are also included, such as viral vectors (for example, replication-defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0085] In a seventh aspect of the embodiments of the present application, a cell is provided, wherein:

[0086] Expressing the specific binding protein of fHbp; or,

[0087] Including the nucleic acid or the vector.

[0088] The term "cell" or "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Easily transformed bacteria include members of the Enterobacteriaceae family, 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.

[0089] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of passages. It should also be understood that all progeny may not be precisely identical in DNA content, due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as that screened for in the originally transformed cell are included. Where a different designation is intended, this is clear from the context.

[0090] In an eighth aspect of the embodiments of the present application, a method for constructing the cell is provided, which comprises the step of introducing the nucleic acid or the vector into the cell to be transformed.

[0091] The method of introduction can be but is not limited to transfection.

[0092] The term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipofection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.

[0093] In a ninth aspect of the embodiments of the present application, a method for producing the fHbp-specific binding protein is provided, wherein the production method uses the cells to produce the fHbp-specific binding protein.

[0094] The specific binding protein can be produced by methods known in the art, including but not limited to hybridoma cells and molecular biology methods. In one embodiment, the present invention screened and obtained a specific monoclonal antibody hybridoma cell line 33G12 based on mice immunized with the fHbp-V2 antigen V2.16, with an antibody titer exceeding 1:10^7.

[0095] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0096] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0097] Example 1

[0098] 1fHbp-V2 recombinant expression and purification

[0099] (1) Based on the amino acid sequence of V2.16 (SEQ ID NO.11), the coding gene was optimized according to the codon preference of E. coli, the gene sequence was synthesized, and the gene encoding V2.16 was constructed into the expression vector pET21b. The restriction sites NdeI / XhoI were introduced to complete the construction of the plasmid. The synthesized plasmid V2.16-pET21b was transformed into E. coli competent cells BL21 (DE3), spread on LB plates containing resistance, and incubated at 37°C. The next day, a single colony was inoculated into LB medium containing the corresponding antibiotics, 1mM IPTG was added, and the cells were induced at 37°C, 180 rpm for 4 hours, and the cells were collected.

[0100] (2) Protein purification: Add lysis buffer (50mM NaH2PO4 / Na2HPO4, 150mM NaCl, pH7.4) to the above bacteria, disrupt the bacteria, and collect the supernatant by centrifugation. In the first step of affinity chromatography, pack the column with 5mL Ni-IDA filler and equilibrate the column with 5 column volumes of binding buffer (50mM NaH2PO4 / Na2HPO4, 150mM NaCl, 20mM imidazole, pH7.4). After loading the sample, perform linear elution, collect the elution fractions according to the absorbance peak, and identify them by SDS-PAGE. Change the buffer, and replace the fraction containing the target protein into anion exchange binding buffer (20mM Tris-HCl, pH8.0) by dialysis. In the second step of anion exchange, the anion exchange column was equilibrated with 5 column volumes of binding buffer. After loading the sample, the flow-through fractions were collected and linearly eluted with elution buffer (20 mM Tris-HCl, 500 mM NaCl, pH 8.0). The eluted fractions were collected according to the peak absorbance and identified by SDS-PAGE (see Figure 1 ), the purity of the target protein was detected by HPLC, and finally V2.16 with a purity of more than 99% was prepared.

[0101] 2 Animal immunization

[0102] V2.16 was mixed with Freund's complete adjuvant in equal volumes, emulsified thoroughly, and injected subcutaneously at multiple sites into 4-6 week old female BALB / c mice at 100 μg / mouse. Two weeks later, a second immunization was performed using the same dose of antigen mixed with Freund's incomplete adjuvant in equal volumes. Blood was collected two weeks after immunization, and 96-well plates were coated with V2.16 protein. Serum was assayed by ELISA. Mice with antibody titers exceeding 1:10,000 were selected and given a 50 μg booster immunization via the tail vein.

[0103] 3. Cell Fusion

[0104] (1) Prepare mouse myeloma SP / 0 cells at least one week before fusion, adjust the cell state to the logarithmic growth phase, select the mouse with the highest ELISA titer, and boost the mice 3 days before fusion with 50 μg / mouse via intraperitoneal injection.

[0105] (2) Fusion: The spleen of the mouse was harvested and mechanically crushed to collect spleen cells, which were filtered through a 200-mesh sieve and washed three times with PBS. SP2 / 0 cells were collected and washed three times with PBS. After counting the cells, the cells were mixed at a ratio of SP / 0: spleen cells = 1:2.5. After centrifugation, the PBS was discarded and the cells were fused using an electrofusion instrument. After the fusion was completed, the cells were centrifuged and complete culture medium containing HAT was added. The cells were resuspended and mixed and then plated in a 96-well plate.

[0106] (3) Medium change: A full medium change is performed 5 days after fusion.

[0107] 4. Hybridoma cell screening

[0108] (1) ELISA test: Coat the immunogen (V2.16), and aspirate the cell culture supernatant for ELISA test 7 days after fusion. Record the positive wells, mark them, and change the medium halfway.

[0109] (2) ELISA retest and screening: The next day, retest the positive wells after the liquid change and screen the coated screening plate, record the required clone number, and perform subcloning.

[0110] 5 Establishment of stable cell lines

[0111] (1) First subcloning: Select the retested positive cell wells from the above for subclone screening, using the limiting dilution method, complete medium, and plate one cell per well in a 96-well plate;

[0112] (2) After 7 days, observe under a microscope, mark the monoclonal wells, perform ELISA testing, discard the clones that turn negative, and for the positive clones, select the wells with vigorous growth and perform a half-fluid change. Re-inspect and reverse screening the next day.

[0113] (3) Secondary subcloning: Select positive clones from the previous step for secondary subclone screening using the limiting dilution method with complete medium and plate the cells in a 96-well plate at a rate of 1 cell per well.

[0114] (4) After 7 days, observe under a microscope, mark the single clone wells and register them, perform ELISA test on the registered clones, and after multiple subcloning until the ELISA test positive rate reaches 100%, select the wells with vigorous growth for expansion culture and freeze preservation.

[0115] The hybridoma cell line finally determined and the monoclonal antibody produced subsequently were both designated as 33G12.

[0116] 6 Epitope Addition Experiment

[0117] (1) Supernatant Titer Experiment: Coat V2.16 protein with CBS at a final concentration of 1 μg / mL, 100 μL / well. After incubating overnight at 4°C, block with 0.1% casein blocking solution for 1 h at room temperature. Wash the plate, add the cell culture supernatant of the stable cell line, dilute it 5-fold, and detect the antibody titer by ELISA. Measure the OD reading with an ELISA reader. 450 Reading.

[0118] (2) Coating Concentration Exploration Experiment: Coat V2.16 protein with CBS at final concentrations of: 1 / 0.333 / 0.111 / 0.037 / 0.012 / 0.004 / 0.001 / 0 μg / mL, 100 μL / well. After incubating overnight at 4°C, block with 0.1% casein blocking solution for 1 h at room temperature. Wash the plate, add the cell culture supernatant of the stable cell line, and detect the antibody titer by ELISA. Measure the OD reading with an ELISA reader. 450 Reading.

[0119] (3) Antibody Addition Experiment: Coat V2.16 protein with CBS at a final concentration of 0.025 μm / mL, 100 μL / well. After incubating overnight at 4°C, block with 0.1% casein blocking solution for 1 h at room temperature. Wash the plate, add 100 μL of the supernatant of a single clone per well, and for the addition wells, add 50 μL of each of the two clones per well, and perform checkerboard detection.

[0120] (4) Addition Index AI: The calculation formula for the addition index AI is: AI = (2*A1 + 2) / (A1 + A2) - 1 / A1 + 2, where A1 and A2 are the OD values of the supernatants of two monoclonal cells after mixing. In this experiment, 2 wells were done, so 2*A1 + 2 is the sum of the OD values of the two wells, and A1 / A2 is the OD value of one monoclonal supernatant. The judgment criteria are: AI < 10%, the antigen epitopes are the same; 10% < AI < 50%, the antigen epitopes are similar; AI > 50%, the antigen epitopes are different.

[0121] 7 Preparation of Monoclonal Antibodies

[0122] (1) Resuscitate the stable hybridoma cell line 33G12, passage the cells to the logarithmic growth phase, and intraperitoneally inject 12 - 14 g BALB / c mice pre-injected with liquid paraffin. Inoculate 1×10 6 cells per mouse. 10 days after injection, the abdomen of the mouse is significantly swollen. After sacrificing the mouse, collect the ascites.

[0123] (2) Equilibration of the purification column: Turn on AKTA PURE and charge the UV to the baseline, then zero it. Connect HiTrap Fibro PrismA to the column valve and equilibrate it with the binding buffer until the conductivity parameter and UV parameter are at the baseline.

[0124] (3) After centrifugation, the ascites was filtered through a 0.22 μm pore size filter membrane and injected into the sample loop.

[0125] (4) After injection, use binding buffer to equilibrate to baseline, add elution buffer to collect the purified product, and immediately neutralize it.

[0126] (5) The purified antibody was concentrated by ultrafiltration and finally exchanged into PBS. It was sterile filtered with a 0.22 μm pore size filter membrane in a clean bench and its purity was determined by SDS-PAGE (see Figure 2 ), and its concentration (2 mg / mL) was determined by Nano-500, and stored at -20°C until use.

[0127] After identification, the nucleotide sequence of the DNA encoding the heavy chain variable region of the monoclonal antibody prepared in the present application is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO.2, and the nucleotide sequence of the DNA encoding the light chain variable region is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO.4.

[0128] The CDRs of the heavy chain variable region are as follows:

[0129] SEQ ID NO.5: GDSISSGY;

[0130] SEQ ID NO.6: ISYSGST;

[0131] SEQ ID NO.7: ARGGGWDYFDY;

[0132] The CDRs of the light chain variable region are as follows:

[0133] SEQ ID NO.8: SSISSNY;

[0134] SEQ ID NO.9: MTS;

[0135] SEQ ID NO. 10: QQGSSIPRT.

[0136] CDRs follow the IMGT system definition.

[0137] 8 Monoclonal antibody titer detection

[0138] (1) Dilute the V2.16 protein prepared in step 1 with CBS and coat the plate at a concentration of 1 μg / mL, 100 μL / well, incubate at 4°C overnight, wash, add 100 μL / well of 0.1% casein-PBS for blocking, incubate at 37°C for 1 hour, and then wash the plate.

[0139] (2) The initial dilution of the monoclonal antibody prepared above was 1:2000, and the monoclonal antibody was serially diluted 3-fold. The plate was incubated at 37°C for 1 hour and then washed.

[0140] (3) Add AP-labeled goat anti-mouse IgG secondary antibody (1:3000 dilution), incubate at 37°C for 1 h, and then wash the plate.

[0141] (4) Add colorimetric solution, incubate at 37°C for 5 min, add stop solution, and read at 405 nm.

[0142] 9 Characterization of Monoclonal Antibodies

[0143] (1) Coat with multiple antigens (including V2.16 and V1.13 and E. coli host proteins) at a concentration of 1 μg / mL, 100 μL / well, incubate at 4°C overnight, wash, add 100 μL / well of 0.1% casein-PBS for blocking, block at 37°C for 1 hour, and then wash the plate.

[0144] (2) The monoclonal antibody prepared above was diluted 3-fold starting at a dilution of 1:2000, incubated at 37°C for 1 hour, and then washed.

[0145] (3) Use goat anti-mouse IgG-AP as the detection antibody, PNPP as the color development, and read at 405 nm.

[0146] Table 1. Potency detection and specific binding identification of anti-fHbp-V2 monoclonal antibodies

[0147]

[0148]

[0149] 10 Flow cytometry detection of fHbp expression abundance

[0150] (1) Resuscitate the strain to be tested, spread an appropriate amount of glycerol on a blood plate, and incubate in a 37°C, 5% CO2 incubator for 16 h;

[0151] (2) Transfer to fresh Mueller-Hinton medium and culture at 37°C in a shaking incubator until OD 600 0.5-0.55;

[0152] (3) Collect 2 mL of bacteria by centrifugation at 12,000 rpm, resuspend in PBS buffer containing 1% paraformaldehyde, and place at 4°C for 30 min. A negative control was set for each bacterial strain.

[0153] (4) Wash the fixed bacteria twice with PBS containing 1% BSA, add the diluted monoclonal antibody prepared above, and incubate at 37°C for 1 h;

[0154] (5) Wash the fixed bacteria twice with PBS containing 1% BSA, add biotinylated goat anti-mouse IgG, and incubate at 37°C for 30 min;

[0155] (6) Wash the fixed bacteria twice with PBS containing 1% BSA, add streptavidin-PE, and incubate at 37°C for 30 min;

[0156] (7) Collect the precipitate by centrifugation, resuspend it in 0.5 mL of PBS, and transfer it to a flow cytometry tube;

[0157] (8) Immediately perform flow cytometry testing.

[0158] Table 2. Analysis of fHbp-V2 expression abundance in serogroup B meningococcal strains

[0159]

[0160] 11 Establishment of antigen quantitative detection method

[0161] The method was established by antibody pairing and condition optimization. The monoclonal antibody 33G12 prepared above was used as the coating antibody, and another universal monoclonal antibody-HRP was used as the detection antibody. The specific operation was as follows:

[0162] (1) Dilute the coating antibody 33G12 to 10 μg / mL with 1*CBS, seal and incubate at 4°C overnight, wash three times with PBST, and pat clean;

[0163] (2) Add 4% BSA blocking solution to the plate, 200 μL / well, incubate at 37°C for 2 h, wash three times with PBST, and tap clean;

[0164] (3) Antigen V2.16 was serially diluted (0, 10, 50, 100, 200, 400, 800, 1600 ng / mL). Two replicates were prepared for each sample. The diluted antigen was added and incubated at 37°C for 1 h. The cells were washed three times with PBST and patted clean.

[0165] (4) Add HRP-labeled universal monoclonal antibody (1:3000 dilution) at 100 μL / well, incubate at 37°C for 1 h, wash three times with PBST, and pat clean;

[0166] (5) Add TMB colorimetric solution (100 μL / well) and incubate at 37°C in the dark for 5-15 min. Immediately add 50 μL 2M H2SO4 or 1M HCl to terminate the reaction, and read the value at 450 nm.

[0167] The detection antibody, namely the universal monoclonal antibody-HRP, is commercially available, and the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO. 12 and SEQ ID NO. 13, respectively.

[0168] Method validation:

[0169] (1) Specificity verification: Coat with monoclonal antibody 33G12 (diluted to a final concentration of 10 μg / mL with 1× CBS), seal and incubate overnight at 4°C. The standard antigen is V2.16 (concentration gradient: 0-5 μg / mL, 12 gradients), and the other antigens are V1.13 and E. coli host protein. The concentration gradients of these antigens are all 0-5 μg / mL. The rest of the method is the same as the specific operation after the method is established. Figure 4 .

[0170] (2) Sensitivity and precision verification: Sensitivity was determined based on the standard curve (see Figure 3 ) to obtain its linear range and minimum detection limit. Precision includes

[0171] a. Repeatability: The precision of the results obtained by the same analyst under the same conditions.

[0172] b. Reproducibility: the precision of the results measured by different laboratories and different analysts;

[0173] c. Intermediate precision: The precision of the results obtained by different analysts using different equipment in the same laboratory at different times; and within the specified range, it is evaluated using at least 6 measurement results and expressed as relative standard deviation (RSD).

[0174] (3) Accuracy verification: Prepare V2.16 standard curve solution; select three points (high, medium, and low concentrations) within the linear range; set up three groups of spike recovery, each with three parallels, all at 100 μL / well.

[0175] Determination of fHbp antigen content in 12B group meningococcal multicomponent vaccine

[0176] (1) Screening of desorption solutions: EDTA buffer, phosphate buffer, trisodium citrate buffer, and diethanolamine buffer were selected for comparison. The above-established antigen quantification method was used to compare the effects of these desorption solutions on the recovery rate of fHbp antigen in fHbp antigen concentrate, vaccine semi-finished products, and finished products, in order to screen out the appropriate desorption solution.

[0177] (2) Screening of desorption temperature and method: Compare the effects of different desorption temperatures (4°C, 25°C, and 37°C) and different desorption methods (water bath and air bath) on the recovery rate of fHbp antigen in fHbp antigen concentrate, vaccine semi-finished products, and finished products, so as to screen out the appropriate desorption temperature and method.

[0178] (3) Using the above-established antigen quantitative detection method, the semi-finished and finished products containing fHbp vaccine were incubated in trisodium citrate buffer in a 37°C water bath, and the obtained fHbp antigen (V2.16) detection rates were in the range of 80-120%.

[0179] Table 3. Antigen quantitative detection method conditions and background value, maximum value and linear range

[0180]

[0181] Table 4. Repeatability verification of antigen quantitative detection method

[0182]

[0183]

[0184] Table 5. Intermediate precision verification of antigen quantitative detection method

[0185]

[0186] SEQ ID NO.1 (heavy chain variable region):

[0187] GAGGTGCAGCTGGAGGAGTCAGGACCTAGCCTCGTGAAAACCTTCTCAGACTCTGTCCCTCACCTGTT

[0188] CTGTCACTGGCGACTCCATCTCCAGTGGTTACTGGAACTGGATCCGGAAATTCCCAGGGAATAAACTT

[0189] GAGTACATGGGTTACATAAGCTACAGTGGAAGCACTTACTACAATCCATCACTCAGAAGTCGAATCTC

[0190] CATCACTCGAGACACATCCAAGAACCAGTACTACCTGCAGTTGAATTCTGTGACTACTGAGGACACA

[0191] GCCACATATTACTGTGCAAGAGGAGGGGGCTGGGACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA。

[0192] SEQ ID NO.2 (Heavy chain variable region):

[0193] EVQLEESGPSLVKPSQTLSLTCSVTGDSISSGYWNWIRKFPGNKLEYMGYISYSGSTYYNPSLRSRISITRD TSKNQYYLQLNSVTTEDTATYYCARGGGWDYFDYWGQGTTLTVSS。

[0194] SEQ ID NO.3 (Light chain variable region):

[0195] CAAATTGTTCTCACCCAGTCTCCAACCACCATGGCTGCATCTCCCGGGGAGAAGATCACTATCACCTG

[0196] CAGTGCCAGCTCAAGTATAAGTTCCAATTACTTCCATTGGTATCAGCAGAAGCCAGGATTCTCCCCTAA

[0197] ACTCTTGATTTATATGACATCCAATCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGG

[0198] GACCTCTTACTCTCTCACAATTGGCACCATGGAGGCTGAAGATGTTGCCACTTACTACTGCCAGCAGGGTAGTAGTATACCACGCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA。

[0199] SEQ ID NO.4 (Light chain variable region):

[0200] QIVLTQSPTTMAASPGEKITITCSASSSISSNYFHWYQQKPGFSPKLLIYMTSNLASGVPARFSGSGSGTSYS LTIGTMEAEDVATYYCQQGSSIPRTFGGGTKLEIK。

[0201] SEQ ID NO.11:

[0202] VAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR

[0203] FDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGK

[0204] AEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAKQ*.

[0205] SEQ ID NO.12:

[0206] EVQLQQPGSELVRPGASVKLSCKASGYTFTSYWMHWVKQRHGQGLEWVGNVYPGSGTTYHGEKFKSKVTLTVDTSSTIAYMHLSSLTSEDSAVYYCTRGWLPYHFDYWGQGTTLTVSS.

[0207] SEQ ID NO.13:

[0208] DILMTQSPASLSMAMGEKVTIRCITSTDIGDDMNWYQQKPGEPPKLLISEGNSLRPGVPSRFSSSGYGTDF VFTIENMLSEDIADYYCLQSDNLPYTFGGGTRLEIK.

[0209] The various technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the various technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of the description in this specification.

[0210] The above-described embodiments only express several implementation methods of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that, for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of the present application shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A specific binding protein of fHbp, characterized in that: The specific binding protein has an antigen binding domain, wherein the antigen binding domain has HCDR1 to HCDR3 shown in SEQ ID NOs. 5 to 7, and LCDR1 to LCDR3 shown in SEQ ID NOs. 8 to 10.

2. The fHbp-specific binding protein according to claim 1, characterized in that: The specific binding protein is an antibody, an antigen-binding fragment of an antibody, or a small module immune drug; Optionally, the specific binding protein is a monoclonal antibody, a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a Fv fragment, a scFv fragment, a linear antibody, a multispecific antibody, a mini antibody, a chelated recombinant antibody, an internal antibody, a binding domain immunoglobulin fusion protein, a small module immune drug, a camelized antibody or an antibody containing VHH.

3. The fHbp-specific binding protein according to any one of claims 1 to 2, characterized in that: The species origin of the heavy chain framework region and the light chain framework region of the antigen binding domain are independently cattle, horses, pigs, sheep, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese or humans.

4. The fHbp-specific binding protein according to claim 3, characterized in that: The antigen binding domain has a heavy chain variable region shown in SEQ ID NO.2 and a light chain variable region shown in SEQ ID NO.

4.

5. The fHbp-specific binding protein according to any one of claims 1 to 2 and 4, characterized in that: The specific binding protein also has a heavy chain constant region and / or a light chain constant region; Optionally, the sequences of the heavy chain constant region and the light chain constant region are each independently selected from the sequence of any one of the constant regions of IgG, IgA, IgM, IgE and IgD.

6. The fHbp-specific binding protein according to claim 5, characterized in that: The species origin of the heavy chain constant region and the light chain constant region are independently cattle, horses, pigs, sheep, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese or humans.

7. Use of the fHbp-specific binding protein according to any one of claims 1 to 6 in the preparation of a detection kit.

8. A detection kit, characterized in that: The detection kit comprises the fHbp-specific binding protein according to any one of claims 1 to 6.

9. The detection kit according to claim 8, characterized in that The detection kit also includes other types of fHbp-specific binding proteins.

10. A method for detecting fHbp or a strain expressing fHbp, characterized in that: The detection method uses the specific binding protein according to any one of claims 1 to 6 or the detection kit according to any one of claims 8 to 9 to detect fHbp or a strain expressing the same.

11. The method for detecting fHbp or a strain expressing the fHbp according to claim 10, characterized in that: The fHbp belongs to the A subfamily.

12. The method for detecting fHbp or a strain expressing the fHbp according to claim 11, characterized in that: The fHbp includes one or more of fHbp variant 2 and fHbp variant 3.

13. A nucleic acid, characterized in that The nucleic acid encodes the fHbp-specific binding protein according to any one of claims 1 to 6.

14. The nucleic acid according to claim 13, characterized in that The nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID NO.1, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in SEQ ID NO.

3.

15. A carrier, characterized in that The vector comprises the nucleic acid of any one of claims 13 to 14.

16. The carrier according to claim 15, characterized in that The vector is a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus or a mammalian cell virus.

17. A cell, characterized in that The cell: Expressing a specific binding protein of fHbp according to any one of claims 1 to 6; or, The method comprises the nucleic acid according to any one of claims 13 to 14 or the vector according to any one of claims 15 to 16.

18. The cell according to claim 17, characterized in that The cells are CHO cells, COS cells, NSO cells, HeLa cells, BHK cells or HEK293 cells.

19. A method for constructing a cell according to any one of claims 17 to 18, characterized in that: The construction method comprises the step of introducing the nucleic acid according to any one of claims 13 to 14 or the vector according to any one of claims 15 to 16 into the cell to be transformed.

20. A method for producing a specific binding protein of fHbp according to any one of claims 1 to 6, characterized in that: The production method uses the cell according to any one of claims 17 to 18 to produce the specific binding protein of fHbp.

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