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 specific binding protein has pertinence to group B meningococcus fHbp-V1, has no cross recognition reaction with A subgroup, does not recognize Escherichia coli host protein, can be used for qualitative and quantitative detection of antigens, and has a good application prospect. The method can be used for identifying, qualitatively and quantitatively detecting the fHbp-V1 antigen in a multi-component group B meningococcus vaccine containing the fHbp-V1 antigen (the accuracy is controlled to be 90-105%), and can also be used for identifying V1 expression abundance of a meningococcus strain.

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 meningitides group B, commonly known as group B meningococcus, can cause epidemic cerebrospinal meningitis and sepsis, which has a high morbidity and mortality rate. It is a common serogroup of invasive cerebrospinal meningitis. At present, group B meningitidis has become the main epidemic strain, and the cases caused by it, group A and group C meningococci account for more than 90% of the total cases.

[0003] Different from other meningococci, the capsular polysaccharide component of group B meningococci is a homologous polymer of α(2-8)N-acetylneuraminic acid (polysialic acid) and an autoantigen, which is similar to α2-8 sialylated human glycoproteins, such as adhesion molecules of neural cells in the fetus. Similarly, the lipopolysaccharide structure of many meningococci has a terminal N-lactose structure similar to the lipose found in human cells, which has the risk of inducing autoimmune diseases, so the outer membrane protein of group B meningitidis has become a candidate antigen for the vaccine. Among them, outer membrane lipoprotein 2086, namely LP2086, can bind to human factor H and downregulate the bypass pathway of the complement activation pathway, so that the bacteria can escape the body's recognition and attack and survive. It is now called factor H binding protein (fHbp).

[0004] fHbp is a specific lipoprotein expressed on the surface of almost all Neisseria meningitidis, with a relative molecular mass of about 29kDa. fHbp is divided into three variants, variant 1 (V1), variant 2 (V2), and variant 3 (V3). V1 is also called subfamily B, and V2 and V3 belong to subfamily A. At present, V1 and V2 strains dominate group B meningococci at home and abroad. According to the comparison of fHbp amino acid sequences, the amino acid sequence of fHbp is relatively conservative, and different subvariants in the same subfamily have 91.6% to 100% homology, and the homology of different subfamilies is about 62.8%.

[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 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 of fHbp and a production method and application thereof, including the following technical solutions:

[0007] One or more embodiments of the present application provide a specific binding protein of 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 module 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 has 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 the preparation of 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 also includes 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 detection 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 the B subfamily.

[0020] In some embodiments of the present application, the fHbp includes fHbp variant 1.

[0021] One or more embodiments of the present application provide a nucleic acid encoding the specific binding protein of the 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, which includes 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 comprises 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 specific binding protein of fHbp, wherein the production method uses the cells to produce the specific binding protein of fHbp.

[0031] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. 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 completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0033] Figure 1 For SDS-PAGE identification of fHbp-V1;

[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] The present application will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and are not used to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms, is not limited to the embodiments and examples described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the present application, and the equivalent form obtained also falls within the protection scope of the present application. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present application, and it should be understood that the present 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 belongs. The terms used herein in the specification of this application are only for the purpose of describing implementation modes and embodiments 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] As used herein, the terms "and / or", "or / and", and "and / or" cover any one of two or more of the related listed items, as well as any and all combinations of the related listed items. The said any and all combinations include combinations of any two of the related listed items, any more of the related listed items, or all of the 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, this technical solution undoubtedly includes the technical solution connected by "logical AND", and undoubtedly also includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example is the technical solution of "A, and / or, B, and / or, C, and / or, D", which includes any one of A, B, C, and D (i.e., the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, combinations of any two or any three of A, B, C, and D, and also includes the combination of the four items A, B, C, and D (i.e., the technical solution connected by "logical AND").

[0042] In this application, terms such as "multiple", "multiple types", "multiple times", and "multiple elements", without special limitation, refer to a quantity greater than or equal to 2. For example, "one or more types" means one type or two or more types.

[0043] As used herein, "its combination", "any combination thereof", "any combination mode thereof", etc. include all suitable combination modes of any two or more of the listed items.

[0044] In this document, the "suitable" in "suitable combination mode", "suitable mode", "any suitable mode", etc. is subject to being able 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] In this document, "preferred", "better", "more preferable", and "it is advisable" are only used to describe the implementation manners or embodiments with better effects, and it should be understood that they do not constitute a limitation on the protection scope of this application.

[0046] In this application, "further", "even further", "especially", etc. are used for descriptive purposes, indicating differences in content, but should not be construed as a limitation on the protection scope of this application.

[0047] In this application, "optionally", "optional", and "optional" mean that it can be either present or absent, that is, it refers to any one of two parallel options: "present" or "absent". If "optional" appears multiple times in a technical solution, without special explanation and without contradictions or mutual constraints, each "optional" is independent.

[0048] In this application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", 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", "fourth", etc. only serve the purpose of non-exhaustive enumerative description and should be understood not to constitute a closed limitation on quantity.

[0049] In this application, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, and also includes an open technical solution containing the listed features.

[0050] In this application, regarding the numerical interval (i.e., the numerical range), unless otherwise specified, the optional numerical values are considered continuous within the above numerical interval, and include the two numerical endpoints (i.e., the minimum value and the maximum value) of this numerical range, 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 this numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer. For example, t is an integer selected from 1 to 10, which means that t is any integer selected from the integer group composed of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when providing multiple range descriptions for features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this article should be understood to include any and all sub-ranges subsumed therein.

[0051] For the temperature parameter in this application, unless otherwise specified, it allows both constant temperature treatment and variation within a certain temperature range. It should be understood that the so-called constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. It is allowed to fluctuate within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C.

[0052] In this application, %(w / w) and wt% both represent 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 with all contents and all purposes. When the 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 the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of 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 modified adaptively according to the description of 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-V1, has no cross-recognition reaction with the A 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-V1 antigens in multi-component group B meningococcal vaccines containing fHbp-V1 antigens (with an accuracy controlled at 90%-105%), and can also be used to identify the expression abundance of V1 in meningococcal strains.

[0056] It is well known in the art that the binding specificity and affinity of an antibody are mainly determined by the CDR sequence. According to mature and well-known existing technologies, the amino acid sequence of the non-CDR region can be easily changed to obtain a variant with similar biological activity. Therefore, the present invention also includes a "functional derivative" of the binding protein. A "functional derivative" refers to a variant with amino acid substitutions, and a functional derivative retains detectable binding protein activity. A "functional derivative" may include "variants" and "fragments" because they have the same CDR sequence as the binding protein described in the present invention and therefore have similar biological activity.

[0057] The antigen binding domains described herein may comprise one or more substitutions, deletions or insertions of amino acids relative to the above CDR sequences, for example, the number of amino acid insertions, substitutions or deletions does not exceed 3, preferably 1. 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. "Conservative amino acid substitutions" are situations in which one amino acid residue is replaced by an amino acid residue with 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 module immune drug; alternatively, 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. In some embodiments, the antigen binding fragment is derived from a complete antibody molecule, such as a monoclonal antibody. The preparation method is 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 antibodies can be divided into κ or λ light chains. The heavy chains can be divided into μ, δ, γ, α or ε, which define the isotype of the antibody as IgM, IgD, IgG, IgA or IgE, respectively. In the light and heavy chains, the variable region is connected to the constant region by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 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 3 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 3 CDRs and 4 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 antigen binding sites / parts, 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 determination of CDR sequences uses the numbering definitions in the Kabat system.

[0060] The term "monoclonal antibody" or "monoclonal antibody" or "monoclonal antibody composition" refers to an antibody molecule product composed of a single molecule. The monoclonal antibody composition presents 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-V1 specific monoclonal antibody that can only recognize the B subfamily and has no cross-reaction with the A subfamily (V2 and V3). It can be used in scenarios such as strain identification, antigen identification 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 in this application. In contrast, a "monospecific" molecule refers to a molecule that specifically binds to a certain target molecule, especially a certain epitope on a certain target molecule, such as a monoclonal antibody that binds to CD3 in this application.

[0062] "Nanoantibodies" are generally defined as in WO 2008 / 020079 or WO 2009 / 138519, and in a specific aspect generally represent VHH, humanized VHH or camelized VH (such as camelized human VH), or generally represent sequence-optimized VHH (e.g., 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 there are mainly 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 IgNAR 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 affinity and solubility are greatly reduced. In one example, the specific binding protein of 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 animals. Their molecular weight is as low as 12-15 kDa, which is only 1 / 10 of that of traditional antibodies. They are the smallest functional single-domain antibodies 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, easy expression and easy engineering transformation, nanobodies can be used as affinity capture reagents, biosensors, in vivo imaging tracers, etc., and have broad application prospects in scientific research, material 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 the percentage of amino acid sequence identity, 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 the parameters suitable for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0066] In some embodiments of the present application, the specific binding protein further has 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 mean regions of antibody variable domains excluding those regions 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 / VH of the heavy and light chains can be obtained by connecting the following numbered CDRs and FRs in the following combinations: 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 human-derived antibody framework region sequences. For example, one or more (e.g., one, two, three, four, five, or six) framework regions of the 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 bring 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 can 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 can be replaced with a human constant region. Relative to non-chimeric antibodies, chimeric antibodies are generally less immunogenic to humans, and therefore may provide 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 the preparation of a detection kit.

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

[0074] In some embodiments of the present application, the detection kit also includes 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, wherein the detection 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 the B subfamily.

[0077] In some embodiments of the present application, the fHbp includes fHbp variant 1.

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

[0079] The detection kit and detection method of the present application can also detect fHbp or the strain expressing it according to the immunoturbidimetric method.

[0080] According to 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 acid and nucleic acid molecules of the present application mainly refer to separated nucleic acid molecules. "Separated" refers to that the molecule is 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 "separated" is not intended to refer to 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 compound 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, 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 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, for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%).

[0083] According to 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 in 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 the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors). Other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. In addition, some vectors can direct the expression of the genes to which they are effectively connected. This vector is referred to herein as a "recombinant expression vector" (or simply "expression vector"). Generally, expression vectors useful in recombinant DNA technology are usually present in the form of plasmids. However, other forms of expression vectors are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses and adeno-associated viruses), which play 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 may include bacteria, microorganisms, plants or animal cells. Easily transformed bacteria include members of the family 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.

[0089] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such names 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 exactly identical in terms of DNA content, due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. Where different names are 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, and the method comprises the step of introducing the nucleic acid or the vector into the cell to be transformed.

[0091] The method of introduction may 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, lipid transfection 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] A ninth aspect of the embodiments of the present application provides a method for producing the specific binding protein of fHbp, wherein the production method uses the cells to produce the specific binding protein of fHbp.

[0094] The present application can produce the specific binding protein by methods known in the art, including but not limited to hybridoma cells and molecular biological methods. In one embodiment, the present application screened and obtained a specific monoclonal antibody hybridoma cell line 62D11 based on fHbp-V1 antigen V1.13 immunized mice, and the antibody titer reached 1:10^7 or above.

[0095] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application 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 manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0096] In the following specific embodiments, 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 test accuracy or operation accuracy are allowed.

[0097] Example 1

[0098] 1. Recombinant expression and purification of fHbp-V1

[0099] (1) According to the amino acid sequence of V1.13 (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 V1.13 was constructed into the expression vector pET21b, and the restriction sites NdeI / XhoI were introduced to complete the construction of the plasmid. The synthesized plasmid V1.13-pET21b was transformed into the competent E. coli cells BL21 (DE3), spread on the LB plate containing resistance, incubated at 37°C, and a single colony was inoculated into the LB medium containing the corresponding antibiotics the next day, 1mM IPTG was added, and the cells were induced at 37°C, 180rpm for 4h, and the cells were collected.

[0100] (2) Protein purification: Add lysis buffer (50 mM NaH 2 PO 4 / Na 2 HPO 4 , 150mM NaCl, pH7.4), disrupt the cells, and collect the supernatant by centrifugation. In the first step of affinity chromatography, 5mL Ni-IDA filler was used to pack the column, and 5 column volumes of binding buffer (50mM NaH 2 PO 4 / Na 2 HPO 4 , 150mM NaCl, 20mM imidazole, pH7.4) to balance the chromatography column. 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 fractions containing the target protein into anion exchange binding buffer (20mM Tris-HCl, pH8.0) by dialysis. In the second step of anion exchange, balance the anion exchange column with 5 column volumes of binding buffer, collect the flow-through fractions after loading the sample, and perform linear elution with elution buffer (20mM Tris-HCl, 500mM NaCl, pH8.0), and collect the elution fractions according to the absorbance peak, and identify them by SDS-PAGE (see Figure 1 ), the purity of the target protein was detected by HPLC, and finally V1.13 with a purity of more than 99% was prepared.

[0101] 2 Animal immunization

[0102] V1.13 was mixed with Freund's complete adjuvant in equal volume ratio, and after sufficient emulsification, 100 μg / mouse was injected subcutaneously at multiple points into 4-6 week old female BALB / c mice. Two weeks later, the same dose of antigen was mixed with Freund's incomplete adjuvant in equal volume ratio, and the second immunization was performed at the same dose. Blood was collected 2 weeks after immunization, and 96-well plates were coated with V1.13 protein. The serum was tested by ELISA, and mice with antibody titers higher than 1:10000 were selected, and 50 μg was boosted by tail vein immunization.

[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 by intraperitoneal injection.

[0105] (2) Fusion: Mouse spleen was obtained, spleen cells were collected after mechanical disruption, filtered through a 200-mesh sieve, washed three times with PBS, SP2 / 0 cells were collected, washed three times with PBS, and the cells were counted and mixed at a ratio of SP / 0: spleen cells = 1:2.5. PBS was discarded after centrifugation, and cell fusion was performed using an electric fusion instrument. After fusion was completed, the cells were centrifuged and complete medium containing HAT was added. The cells were resuspended and mixed and then plated in a 96-well plate.

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

[0107] 4. Hybridoma Cell Screening

[0108] (1) ELISA test: Coat the immunogen (V1.13), 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 re-examination and screening: The next day, re-examine the positive wells after the liquid change and screen the coated screening originals, 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, use the limiting dilution method, complete medium, and plate 1 cell per well in a 96-well plate;

[0112] (2) After 7 days, observe under a microscope, mark the single clone wells, perform ELISA test, discard the clones that turn negative, and select the wells with vigorous growth for half-fluid change for the positive clones. Re-test 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 and 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 is 100%, select the wells with vigorous growth for expansion culture and cryopreservation.

[0115] Both the finally determined hybridoma cell line and the subsequent monoclonal antibodies produced are denoted as 62D11.

[0116] 6 Epitope addition experiment

[0117] (1) Supernatant titer experiment: Coat V1.13 protein with CBS at a final concentration of 1 μg / mL, 100 μL / well, incubate overnight at 4°C, then 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 5-fold, detect the antibody titer by ELISA, and measure the OD reading with an enzyme-linked immunosorbent assay reader. 450 Reading.

[0118] (2) Coating concentration exploration experiment: Coat V1.13 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, incubate overnight at 4°C, then 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, detect the antibody titer by ELISA, and measure the OD reading with an enzyme-linked immunosorbent assay reader. 450 Reading.

[0119] (3) Antibody addition experiment: Coat V1.13 protein with CBS at a final concentration of 0.025 μm / mL, 100 μL / well, incubate overnight at 4°C, then block with 0.1% casein blocking solution for 1 h at room temperature, wash the plate, add 100 μL / well of single clone supernatant, and for the addition wells, add 50 μL / well of each of two clones, 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 is the OD value of the mixture of two monoclonal cell supernatants. 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 62D11, passage the cells to the logarithmic growth phase, intraperitoneally inject 12 - 14 g BALB / c mice pre-injected with liquid paraffin, inoculate 1×10 6 cells per mouse. Ten days after injection, the mouse peritoneal cavity is significantly swollen. After sacrificing the mouse, collect the ascites.

[0123] (2) Balance the purification column: Turn on the AKTA PURE and charge the UV to the baseline and adjust to zero. Connect the HiTrap Fibro PrismA to the column valve and use the binding buffer to balance until the conductivity and UV parameters 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 balance to baseline, add elution buffer to collect the purified product, and neutralize it immediately.

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

[0130] SEQ ID NO.6: IFPRSGTT;

[0131] SEQ ID NO.7: NYGYAWFPY;

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

[0133] SEQ ID NO.8: HSISNN;

[0134] SEQ ID NO.9: YAS;

[0135] SEQ ID NO.10:QQSSSWPYT;

[0136] The above CDRs follow the IMGT system definition.

[0137] 8 Monoclonal antibody titer detection

[0138] (1) V1.13 protein was diluted with CBS and coated at a concentration of 1 μg / mL, 100 μL / well, incubated at 4°C overnight, and after washing, 100 μL / well of 0.1% casein-PBS was added for blocking. The plate was washed after blocking at 37°C for 1 h.

[0139] (2) The initial dilution of the monoclonal antibody prepared above was 1:2000, and then the monoclonal antibody was 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 V1.13 and V2.16 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 h, and then wash the plate.

[0144] (2) The anti-V1.13 monoclonal antibody prepared above was diluted 3-fold with a starting 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 the value at 405 nm.

[0146] Table 1. Potency detection and specific binding identification of anti-fHbp-V1 monoclonal antibodies

[0147]

[0148]

[0149] 10 Flow cytometry detection of fHbp expression abundance

[0150] (1) Resuscitate the strain to be tested, take an appropriate amount of glycerol and spread it on a blood plate, and incubate at 37°C and 5% CO 2 Incubate in an 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) Take 2 mL of each strain and centrifuge at 12000 rpm to collect the cells, resuspend the cells in PBS buffer containing 1% paraformaldehyde, and place them at 4°C for 30 min. Set up a negative control for each strain.

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

[0154] (5) Wash the fixed bacteria twice with PBS containing 1% BSA, add biotin-labeled 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, add 0.5 mL of PBS to resuspend it, and transfer it to a flow tube;

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

[0158] Table 2. Analysis of fHbp-V1 expression abundance in group B meningococcal strains

[0159]

[0160] 11 Establishment of quantitative antigen detection method

[0161] The method establishment includes antibody pairing and condition optimization, using the above-prepared anti-V1.13 monoclonal antibody 62D11 as the coating antibody and another universal monoclonal antibody-HRP as the detection antibody. The specific operation is:

[0162] (1) Dilute the coating antibody 62D11 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) The antigen V1.13 was diluted in series (0, 10, 50, 100, 200, 400, 800, 1600 ng / mL), with two replicates 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 tapped clean.

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

[0166] (5) Add TMB colorimetric solution (100 μL / well), incubate at 37°C in the dark for 5-15 min, and immediately add 50 μL 2M H 2 SO 4 Or 1M HCl to terminate the reaction, and then read the value at 450nm.

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

[0168] Method Validation:

[0169] (1) Specificity verification: Coat with monoclonal antibody 62D11 (diluted to a final concentration of 10 μg / mL with 1×CBS), seal and incubate overnight at 4°C. V1.13 (concentration gradient: 0-1 μg / mL, 12 gradients) was used as the standard antigen, and V2.16 and E. coli host protein were used as the other antigens. The concentration gradients of these antigens were 0-1 μg / mL. The rest of the methods were the same as the specific operations after the method was established. Figure 4 .

[0170] (2) Sensitivity and precision verification: Sensitivity was measured according to 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 V1.13 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 effects of these desorption solutions on the recovery rates of fHbp antigen in fHbp antigen stock solutions, vaccine semi-finished products and finished products were compared using the established antigen quantification method 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 stock solution, vaccine semi-finished products and finished products, so as to screen out the appropriate desorption temperature and method.

[0178] (3) Using the antigen quantitative detection method established above, the semi-finished and finished products containing fHbp vaccine were incubated in trisodium citrate buffer at 37°C in a water bath, and the obtained fHbp antigen (V1.13) 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] CAGGTGCAGCTGAAGCAGTCTGGAGCTGAGCTGGCGAGTCCCGGGGCTTCAGTGAAGCTGTCCTGC

[0188] AAGACTTCTGGCTACACCTTCACTGACTACTATTTAAACTGGGTGAGGCAGAGGACTGGACAGGGCC

[0189] CTGAGTGGATTGGAGAGATTTTTCCTAGAAGTGGTACAACTTACTACAATAAGAAATTCGAGGGCAAG

[0190] GCCATACTGACTGCAGACACATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGG

[0191] ACTCTGCTGTCTACTTCTGTAACTACGGCTACGCCTGGTTTCCTTACTGGGGCCAAGGGACACTGGTCACTGTCTCTGCA。

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

[0193] QVQLKQSGAELASPGASVKLSCKTSGYTFTDYYLNWVRQRTGQGPEWIGEIFPRSGTTYYNKKFEGKAIL TADTSSSTAYMQLSSLTSEDSAVYFCNYGYAWFPYWGQGTLVTVSA。

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

[0195] GATATTGTGCTAACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGCGTCAGTCTTTCCTGC

[0196] AGGGCCAGCCACAGTATTAGCAACAACCTCCACTGGTATCAACTTAAATCACATGAGTCTCCAAGGCT

[0197] TCTCATCAAGTATGCTTCGCAGTCCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATCAGGGA

[0198] CAGATTTCACCCTCAGTATCAACAGTGTGGAGACTGAAGATTTTGGAATGTATTTCTGTCAACAGAGTAGCAGCTGGCCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA。

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

[0200] DIVLTQSPATLSVTPGDSVSLSCRASHSISNNLHWYQLKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTTLSI NSVETEDFGMYFCQQSSSWPYTFGGGTKLEIK.

[0201] SEQ ID NO.11:

[0202] VAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR

[0203] FDFIRQIEVDGKLITLESGEFQVYKQSHSALTALQTEQVQDSEDSGKMVAKRQFRIGDIAGEHTSFDKLPK

[0204] GGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVELATAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGQAQEVAGSAEVETANGIHHIGLAAKQ*.

[0205] SEQ ID NO.12:

[0206] EVQLQQPGSELVRPGASVKLSCKASGYTFTSYWMHWVKQRHGQGLEWVGNVYPGSGTTYHGEKFKSKVTLTVDTSSTIAYMHLSSLTSEDSAVYYCTRGWLPYHFDYWGQGTTLTVSS.

[0207] SEQ ID NO.13:

[0208] DILMTQSPASLSMAMGEKVTIRCITSTDIGDDMNWYQQKPGEPPKLLISEGNSLRPGVPSRFSSSGYGTDF VFTIENMLSEDIADYYCLQSDNLPYTFGGGTRLEIK.

[0209] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the 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 this specification.

[0210] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on 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 deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain 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 B subfamily.

12. The method for detecting fHbp or a strain expressing the fHbp according to claim 11, characterized in that: The fHbp includes fHbp variant 1.

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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