Specific binding protein of VZV gE as well as production method and application thereof

Abstract

The invention belongs to the technical field of immunological analysis, and relates to a specific binding protein of VZV gE as well as a production method and application thereof. The specific binding protein has one or more of an antigen binding structural domain a and an antigen binding structural domain b; wherein the antigen binding domain a has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.8 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.9; and the antigen binding domain b has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.10 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.11. By adopting the specific binding protein of the VZV gE provided by the invention, a method for detecting the VZV gE or the mutant thereof, which is high in sensitivity, strong in specificity, simple and convenient to operate and rapid, can be established.

Description

Technical Field

[0001] The present application belongs to the technical field of immunological analysis, and relates to a VZV gE specific binding protein and a production method and application thereof. Background Art

[0002] Varicella-zoster virus (VZV) is an enveloped double-stranded DNA virus of the alphaherpesvirus subgenus of the Herpesviridae family. It has a genome size of 125 kb and only one serotype. Chickenpox caused by the initial infection with VZV is a self-limiting disease, and generally provides lifelong immunity after recovery. However, VZV can remain latent in the dorsal root ganglia of the spinal cord or cranial nerve ganglia, and when reactivated can cause herpes zoster (HZ), an acute infectious skin disease that is common in the elderly, immunocompromised, and immunosuppressed people. In severe cases, it can cause neuralgia or multiple complications.

[0003] Currently, vaccination is the preferred option for the prevention and treatment of herpes zoster. VZV virus expresses at least 70 proteins, among which glycoprotein E (gE) is the most expressed glycoprotein and can induce a strong immune response. It is the main antigen in vaccine research. Therefore, the specific detection of VZV gE antigen is particularly important in VZV vaccine research. Summary of the invention

[0004] Based on this, one or more embodiments of the present application provide a VZV gE specific binding protein and a production method and application thereof, including the following technical solutions:

[0005] One or more embodiments of the present application provide a specific binding protein of VZV gE, wherein the specific binding protein has one or more of an antigen binding domain a and an antigen binding domain b;

[0006] in,

[0007] The antigen binding domain a has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.8 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.9;

[0008] The antigen binding domain b has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.10 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.11.

[0009] 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;

[0010] 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 intrabody, a nanobody, a binding domain immunoglobulin fusion protein, a small module immune drug, a camelized antibody or an antibody containing VHH.

[0011] In some embodiments of the present application, the specific binding protein includes an antibody constant region.

[0012] In some embodiments of the present application, the sequence of the antibody constant region is selected from the sequence of any one of the constant regions of IgG, IgA, IgM, IgE and IgD.

[0013] In some embodiments of the present application, the antibody constant region is derived from cattle, horses, pigs, sheep, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese or humans.

[0014] One or more embodiments of the present application provide a nucleic acid encoding the VZV gE-specific binding protein.

[0015] One or more embodiments of the present application provide a recombinant vector, which includes the nucleic acid.

[0016] In some embodiments of the present application, the recombinant vector is a plasmid, a bacteriophage, a plant cell virus or an animal cell virus.

[0017] One or more embodiments of the present application provide a cell, wherein the cell expresses the specific binding protein of VZV gE, comprises the nucleic acid, or comprises the recombinant vector.

[0018] 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.

[0019] 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 recombinant vector into the host cell to be transformed.

[0020] In some embodiments of the present application, the method of introduction includes transfection.

[0021] One or more embodiments of the present application provide a method for producing the specific binding protein of VZV gE, which comprises using the cells to produce the specific binding protein of VZV gE.

[0022] One or more embodiments of the present application provide a use of the VZV gE specific binding protein in the preparation of a detection kit.

[0023] One or more embodiments of the present application provide a VZV gE detection kit, which includes the VZV gE specific binding protein.

[0024] In some embodiments of the present application, the detection kit comprises an antibody 1 having the antigen-binding domain a and an antibody 2 having the antigen-binding domain b.

[0025] One or more embodiments of the present application provide a method for detecting VZV gE or a mutant thereof in a sample, the detection method comprising:

[0026] The specific binding protein or the detection kit is used to detect VZV gE or its mutant in the sample to be tested.

[0027] In some embodiments of the present application, the amino acid sequence of the mutant is shown as SEQ ID NO.2.

[0028] In some embodiments of the present application, the sample to be tested includes cell fermentation broth.

[0029] 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

[0030] 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.

[0031] Figure 1 Map of the recombinant plasmids constructed for this application. DETAILED DESCRIPTION

[0032] 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.

[0033] 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.

[0034] the term

[0035] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0036] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of 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 that are all connected by "logical and", and undoubtedly includes technical solutions that are all 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 that is all 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 combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0037] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0038] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0039] 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.

[0040] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of this application.

[0041] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0042] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0043] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood 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 enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0044] In the present 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.

[0045] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and 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 1-10, indicating that t is any integer selected from the integer group 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 combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges included therein.

[0046] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0047] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0048] 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.

[0049] In a first aspect of the embodiments of the present application, a specific binding protein of VZV gE is provided, wherein the specific binding protein has one or more of an antigen binding domain a and an antigen binding domain b;

[0050] in,

[0051] The antigen binding domain a has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.8 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.9;

[0052] The antigen binding domain b has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.10 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.11.

[0053] By using the specific binding protein of VZV gE in the examples of the present application, a double antibody sandwich ELISA kit and detection method for detecting recombinant VZV glycoprotein E with high sensitivity, strong specificity, simple operation and rapid detection can be established.

[0054] In the present application, the heavy chain variable region of the antigen binding domain a has the sequence shown in SEQ ID NO.8 or has a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) identical to SEQ ID NO.8.

[0055] In the present application, the light chain variable region of the antigen binding domain a has the sequence shown in SEQ ID NO.9 or has a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) identical to SEQ ID NO.9.

[0056] In the present application, the heavy chain variable region of the antigen binding domain b has the sequence shown in SEQ ID NO.10 or a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) identical to SEQ ID NO.10.

[0057] In the present application, the light chain variable region of the antigen binding domain b has the sequence shown in SEQ ID NO.11 or a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) identical to SEQ ID NO.11.

[0058] 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.

[0059] The term "binding (or binding to)" herein refers to the interaction of an antibody with an epitope of a target protein. The term "epitope" as used herein refers to the dispersed three-dimensional site of an antigen recognized by the antibody of the present application. The antibody of the present application can be incorporated into a pharmaceutical composition, which can be prepared by methods well known in the art and comprises the antibody of the present application and one or more pharmaceutically acceptable carriers and / or diluents (e.g., Remington, The Science and Practice of Pharmacy, 22nd edition, Loyd V., Ed., Pharmaceutical Press, 2012, which provides a compilation of formulation techniques commonly known to practitioners. Carriers suitable for pharmaceutical compositions include any material that can retain molecular activity and is unresponsive to the patient's immune system after binding to the antibody of the present application.

[0060] The specific binding protein of VZV gE of the present application may have the above-mentioned heavy chain variable region and light chain variable region, or may be a derivative fragment thereof, may be a derivative fragment with a changed FR in the variable region, may be a derivative fragment with a changed CDRs, for example, the derivative fragment is formed by amino acid substitution ("conservative modification" or "conservative substitution or replacement") at no more than 6 sites relative to its corresponding CDR, and retains the biological activity consistent with its corresponding complementary determining region. For example, the derivative fragment is substituted at 1, 2, 3, 4, 5 or 6 sites relative to its corresponding complementary determining region, and may be one amino acid replaced by another amino acid, or one amino acid replaced by multiple (for example, 2) amino acids.

[0061] In the CDRs provided in the present application, the derivative fragment (conservative variant) refers to a polypeptide formed by replacing 1, 2, or 3 amino acids with amino acids of similar or similar properties compared to the amino acid sequence of the antibody of the present application. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0062] Table A

[0063] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0064] "Conservative modification" or "conservative substitution or replacement" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), so that changes can be made frequently without changing the biological activity of the protein. Those skilled in the art know that, in general, single amino acid replacements in non-essential regions of a polypeptide do not substantially change the biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). In addition, replacement of amino acids with similar structure or function is unlikely to destroy biological activity.

[0065] In the present application, the specific binding protein is an antibody, an antigen-binding fragment of an antibody or a small modular 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 intrabody, a nanobody, a binding domain immunoglobulin fusion protein, a small modular immune drug, a camelized antibody or an antibody containing VHH.

[0066] The "antibody" mentioned in this application refers to immunoglobulin. A complete antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so its antigenicity is also different. Based on this, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain respectively. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into κ chain or λ chain by the difference in the constant region. Each of the five types of Ig can have κ chain or λ chain.

[0067] The specific binding protein of the present application can be a mouse antibody or a rabbit antibody.

[0068] In some embodiments of the present application, the specific binding protein includes an antibody constant region. The present application does not specifically limit the source of the sequence of the constant region. In some embodiments of the present application, the sequence of the antibody constant region is selected from the sequence of any one of the constant regions of IgG, IgA, IgM, IgE and IgD.

[0069] The present application does not specifically limit the species of the antibody constant region. In some embodiments of the present application, the species of the antibody constant region is cattle, horses, pigs, sheep, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese or humans.

[0070] The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, the antibody binds with an affinity (KD) of about less than 10-8M, such as about less than 10-9M, 10-10M, 10-11M, 10-12M or less.

[0071] The term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Typically, the antibodies of the present disclosure bind to TrkA or its epitope with a dissociation equilibrium constant (KD) of less than about 10-7M, such as less than about 10-8M or 10-9M, for example, in the present disclosure, the affinity of the antibody to the cell surface antigen is determined by FACS to characterize the KD value.

[0072] In a second aspect of the embodiments of the present application, a nucleic acid is provided, wherein the nucleic acid encodes the specific binding protein of VZV gE.

[0073] The term "nucleic acid molecule" as used in this application refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively connected to the coding sequence.

[0074] The nucleic acid molecules of the present application mainly 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, buffer or salts, unless they are present in an amount that significantly interferes with the experimental or therapeutic use of the compounds as described herein.

[0075] According to a third aspect of the embodiments of the present application, a recombinant vector is provided, wherein the recombinant vector comprises the nucleic acid.

[0076] Optionally, the vector is selected from mammalian cell virus, bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus or a combination thereof.

[0077] The term "vector" is also "expression vector" and refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded DNA loop into which another DNA segment can be connected. In another embodiment, the vector is a viral vector, in which another DNA segment can be connected to a viral genome. The vector disclosed herein can be autonomously replicated in a host cell introduced therein (e.g., a bacterial vector and an additional mammalian vector having a bacterial origin of replication) or can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating with the host genome (e.g., a non-additional mammalian vector). On the whole, the vector can be selected from, but is not limited to, a mammalian cell virus, a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, or a combination thereof.

[0078] According to a fourth aspect of the embodiments of the present application, a cell is provided, wherein the cell expresses the specific binding protein of VZV gE, comprises the nucleic acid, or comprises the recombinant vector.

[0079] 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.

[0080] 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.

[0081] The fifth aspect of the embodiments of the present application provides a method for constructing the cell, which comprises the step of introducing the nucleic acid or the recombinant vector into the host cell to be transformed.

[0082] In some embodiments of the present application, the method of introduction includes transfection.

[0083] In a sixth aspect of the embodiments of the present application, a method for producing the specific binding protein of VZV gE is provided, wherein the production method comprises using the cells to produce the specific binding protein of VZV gE.

[0084] The specific binding protein (antibody or antigen binding fragment) of the present application can be prepared and purified by conventional methods. For example, the cDNA sequences encoding the heavy chain and light chain can be cloned and recombined into an expression vector. The recombinant immunoglobulin expression vector can stably transfect host cells. As a more recommended prior art, the mammalian expression system will lead to glycosylation of the antibody, especially at the highly conserved N-terminal site in the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to VZV gE. Positive clones are expanded and cultured in a bioreactor to produce antibodies. The culture fluid that secretes antibodies can be purified by conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing an adjusted buffer. Non-specifically bound components are washed away. The bound antibodies are then eluted using the pH gradient method, and the antibody fragments are detected by SDS-PAGE and collected. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The obtained product needs to be immediately frozen, such as -70°C, or lyophilized.

[0085] Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, such as the Cold Spring Harbor Manual of Antibody Experimental Techniques, Chapters 5-8 and 15. For example, mice can be immunized with human TrkA or fragments thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in the application are genetically engineered to add one or more human FR regions to the non-human CDR regions. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulins, 2001 ISBN012441351.

[0086] In a seventh aspect of the embodiments of the present application, a use of the VZV gE specific binding protein in the preparation of a detection kit is provided.

[0087] In an eighth aspect of the embodiments of the present application, a VZV gE detection kit is provided, wherein the detection kit comprises the VZV gE specific binding protein.

[0088] In some embodiments of the present application, the detection kit comprises an antibody 1 having the antigen-binding domain a and an antibody 2 having the antigen-binding domain b.

[0089] The present application uses the VZV gE specific binding protein in the detection kit to compete with the VZV gE or its mutant in the test sample.

[0090] When the term "competes" is used in the context of antigen binding proteins that compete for the same epitope, it means competition between antigen binding proteins, which is determined by an assay in which the antigen binding protein (e.g., antibody or functional fragment thereof) to be tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen (e.g., TrkA antigen or fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen binding protein competes with another, such as: solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid phase direct label assays, solid phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); using I-1 2 Solid phase direct labeling RIA with 5-label (see, e.g., Morel et al., 1988, Molec. Immunol. 25: 7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176: 546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82). Typically the assay involves the use of purified antigen bound to a solid surface or cells bearing either an unlabeled test antigen binding protein or a labeled reference antigen binding protein. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cells in the presence of the test antigen binding protein. Typically the test antigen binding protein is present in excess. Antigen binding proteins identified by competitive assays (competing antigen binding proteins) include: antigen binding proteins that bind to the same epitope as a reference antigen binding protein; and antigen binding proteins that bind to adjacent epitopes sufficiently close to the binding epitope of the reference antigen binding protein that the two epitopes sterically interfere with each other for binding. Additional details on methods for determining competitive binding are provided in the Examples herein. Typically, when the competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more of the specific binding of the reference antigen binding protein to the common antigen.In certain instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0091] In a ninth aspect of the embodiments of the present application, a method for detecting VZV gE or a mutant thereof in a sample is provided, the detection method comprising:

[0092] The specific binding protein or the detection kit is used to detect VZV gE or its mutant in the sample to be tested.

[0093] In some embodiments of the present application, the amino acid sequence of the mutant is shown as SEQ ID NO.2.

[0094] In some embodiments of the present application, the sample to be tested includes cell fermentation broth.

[0095] The present application relates to a method for immunodetection or determination of a target antigen (e.g., VZV gE or a mutant thereof), a reagent for immunodetection or determination of a target antigen (e.g., VZV gE or a mutant thereof), a method for immunodetection or determination of cells expressing a target antigen (e.g., VZV gE or a mutant thereof), and a diagnostic agent for diagnosing a disease associated with cells positive for the target antigen (e.g., VZV gE or a mutant thereof), which comprises the antibody or antibody fragment of the present application that specifically recognizes the target antigen (e.g., VZV gE or a mutant thereof) and binds to the amino acid sequence of the extracellular region or the three-dimensional structure thereof as an active ingredient.

[0096] In the present application, the method for detecting or measuring the amount of the target antigen (e.g., VZV gE or its mutant) can be any known method. For example, it includes an immunoassay or assay method. An immunoassay or assay method is a method for detecting or measuring the amount of antibody or antigen using a labeled antigen or antibody. Examples of immunoassay or assay methods include radioactive material labeled immunoantibody methods (RIA), enzyme immunoassays (EIA or ELISA), fluorescent immunoassays (FIA), luminescent immunoassays, protein immunoblotting, physicochemical methods, etc.

[0097] In order to detect cells expressing the polypeptide, known immunodetection methods can be used, and preferably immunoprecipitation, fluorescent cell staining, immunohistostaining, etc. In addition, fluorescent antibody staining using FMAT8100 HTS system (Applied Biosystem) can be used.

[0098] 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.

[0099] 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.

[0100] This application involves the following technical solutions:

[0101] 1. Using the CHO eukaryotic expression system to express recombinant VZV glycoprotein E, including: PCR amplification of the target fragment and cloning into the expression vector pcDNA3.1(+), transfecting the successfully constructed recombinant expression plasmid into the CHO expression system for cultivation, and obtaining highly purified recombinant VZV gE through complex cation and molecular sieve chromatography. The main process is as follows:

[0102] (1) The nucleotide sequence of the gene encoding VZV gE (SEQ ID NO. 1, 1872 bp) was obtained from GenBank and analyzed using SignalP software and DNAMAN tools: VZV gE contains a front signal peptide (1-30 aa), a hydrophobic amino acid region (226-240 aa, 480-494 aa), and a transmembrane region (545-561 aa).

[0103] SEQ ID NO.1 (nucleotide sequence of the gene encoding VZV gE, 1872 bp):

[0104]

[0105] (2) Gene editing technology was used to mutate the V231Y and V489Q amino acids in the hydrophobic amino acid region (226-240aa, 480-494aa), cut the transmembrane region (547-561aa) without retaining the amino acids, and cut and modify the extracellular region, with a final length of 544aa. The amino acid sequence of the recombinant VZV gE obtained after editing is shown in SEQ ID NO.2 (544aa).

[0106] SEQ ID NO.2 (amino acid of recombinant VZV gE, 544aa):

[0107] .

[0108] (3) Construction of recombinant plasmid:

[0109] The target fragment gE was obtained by primers VZV-gE-F1 (SEQ ID NO.3: AAGCTTGCCACCATGGGCACCGTGAACAAGC) and VZV-gE-R1 (SEQ ID NO.4: TAGTGGATCCTCATTAGGCCAGGCCGCCGGTCC);

[0110] The linearized vector pcDNA3.1 was obtained by primers pcDNA3.1(+)-F (SEQ ID NO.5:TGAGGATCCACTAGTCCAGT) and pcDNA3.1(+)-R (SEQ ID NO.6:GGTGGCAAGCTTAAGTTTAAACG), and the recombinant plasmid pcDNA3.1(+)-VZV-gE was constructed by homologous recombination. The nucleotide sequence is shown in SEQ ID NO.7 (5428 bp). Figure 1 shown.

[0111] SEQ ID NO.7 (nucleotide sequence of recombinant plasmid pcDNA3.1(+)-VZV-gE) (5428 bp):

[0112]

[0113] 2. Recombinant VZV glycoprotein E was expressed using a CHO eukaryotic expression system and a double antibody sandwich ELISA detection kit was prepared, comprising: (1) a 96-well ELISA plate coated with a monoclonal antibody (capture antibody), (2) a recombinant gE protein rabbit monoclonal antibody (detection antibody), (3) a horseradish peroxidase-labeled antibody (HRP-labeled goat anti-rabbit secondary antibody), (4) a gE antigen standard, (5) an antibody diluent / sample diluent, (6) a single-component TMB colorimetric solution, (7) a stop solution, and (8) a washing solution.

[0114] Preparation of capture antibody: After highly purified, the recombinant VZV gE expressed by the HEK-293F culture system was injected intraperitoneally into immunized mice to obtain serum. The mouse spleen cells were fused with mouse myeloma cells SP2 / 0 by the PEG fusion method and cultured. The antibody-positive wells were subcloned by the limiting dilution method to obtain hybridoma cells. Paraffin oil was injected intraperitoneally into the mice to suppress the immune system of the mice. After 1 week, the hybridoma cell suspension was inoculated intraperitoneally. When the abdominal cavity of the mice was obviously swollen, the ascites was collected with a sterile syringe. The titer of the ascites antibody was detected by indirect ELISA, and the mouse anti-VZV-gE monoclonal antibody was obtained after chromatography purification and used as the capture antibody. The amino acid sequence of the heavy chain variable region of the capture antibody is shown in SEQ ID NO.8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.9.

[0115] SEQ ID NO.8 (amino acid sequence of the heavy chain variable region of the capture antibody):

[0116] QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGPGVGWIRQPSGKGLEWLAHIWWDDVKRGNPALKSRLTISKDTSSSQVFLKIASVDTADTATYYCARIRGTRILPYFDYWGQGTTLTVSS;

[0117] SEQ ID NO.9 (amino acid sequence of the light chain variable region of the capture antibody):

[0118] DIQMTQTTSSLSASLGDRVTISCRASGDISNDLNWYQQKPDGTVKALIYYTSRLVSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWDFGGGTKLEIK.

[0119] Preparation of detection antibody: After highly purified, the recombinant VZV gE expressed in the CHO culture system was subcutaneously injected into New Zealand rabbits for immunization, and the anti-VZV-gE antibody titer of the rabbit serum after immunization was collected and detected. After B cell separation, monoclonal B cell culture, antibody screening and identification, amplification culture and purification, monoclonal antibodies were obtained. The amino acid sequence of the heavy chain variable region of the detection antibody is shown in SEQ ID NO.10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.11.

[0120] SEQ ID NO.10 (amino acid sequence of the heavy chain variable region of the detection antibody):

[0121] EVHLQQSGPELVKPGASVKISCKTSGYTFTWYIMHWVKQSHGKSLEWIGGINPNNGDTSGNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARSGVYFDVYGAQGTLVTVSA.

[0122] SEQ ID NO.11 (amino acid sequence of the light chain variable region of the detection antibody):

[0123] DVAMTQTPLTLSVTIGQPASISCKSGQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSELDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWGGTHFPRTFGGGTKLEIK.

[0124] Enzyme-labeled antibody: Goat Anti-Rabbit IgG, HRP Conjugated (Cat. No.: CW0103S; Specification: 100 μL / vial) was purchased from the supplier Kangwei Century.

[0125] Standard product: Prepare recombinant VZV glycoprotein E stock solution, obtain highly purified recombinant VZVgE by chromatography, and the purity of the purified recombinant truncated gE reaches more than 99% by high performance liquid chromatography.

[0126] Example 1. Preparation of capture antibodies

[0127] The preparation method of monoclonal antibody (capture antibody) comprises the following steps:

[0128] a. Transient transfection of 293F cells to prepare recombinant VZV gE

[0129] The above recombinant plasmid pcDNA3.1(+)-VZV-gE was expressed in HEK-293F suspension cells by transient transfection and further purified.

[0130] ①The HEK-293F cells were subcultured for 3 generations.

[0131] ②Preheat Expi Fectamine at 37℃ TM 293Reagent.

[0132] ③7.5×10 7 cells were added to 27 mL of culture medium (the final concentration of transfection was 2.5 × 10 6 cells / mL).

[0133] ④Prepare two centrifuge tubes A and B, add 30 μg of plasmid, final volume 1.5 mL, mix gently.

[0134] ⑤ Add to centrifuge tube B ExpiFectamine TM 81μL, final volume 1.5mL, gently mix, incubate at room temperature for 5min, then add solution B to solution A, gently mix, let stand at room temperature for 20min, add 3mL of A / B mixture dropwise into 27mL of cell culture medium, and tilt to mix.

[0135] ⑥Then incubate in a shaker incubator at 37°C, 8% CO2, and 120 rpm for 18 h, then add ExpiFectamine TM 293 Transfection Enhancer 1 and ExpiFectamine TM 293 Transfection Enhancer 2, gently shake the culture bottle during the addition process.

[0136] ⑦ Harvest the cell fluid 5 days after transfection, centrifuge at 3000g for 5 min, take the supernatant, harvest the extracellular protein, and store it at -80℃.

[0137] ⑧After column chromatography, highly purified recombinant VZV gE protein was obtained.

[0138] Table 1

[0139]

[0140] b. Immunize animals to obtain serum

[0141] The recombinant VZV gE protein was mixed with Freund's adjuvant in a certain proportion and injected intraperitoneally to immunize BALB / c mice. The immunization dose was 50 μg / mouse. Each injection was given 21 days apart for a total of 3 immunizations. Blood was collected from mice 21 days after the last immunization and serum was obtained by centrifugation at 2000-4000 rpm for 15 min. The anti-VZV gE antibody titer in the serum was detected and confirmed by ELISA.

[0142] c. Preparation of monoclonal antibodies

[0143] Mouse spleen cells were fused with mouse myeloma cells SP2 / 0 by PEG fusion method, and fused cells were selected by HAT-RPMI1640 complete medium. The growth of fused cells was observed daily, and the antibody-positive wells were cultured for 7-10 days. The limited dilution method was used to subclone. After screening, hybridoma cells that can stably secrete anti-VZV gE antibodies were obtained. Mice were intraperitoneally injected with paraffin oil 500μl / mouse to suppress the mouse immune system. One week later, hybridoma cell suspension was inoculated intraperitoneally, and the cell number was 5×10 6 When the abdominal cavity of the mouse was obviously swollen, the ascites was collected with a sterile syringe. The titer of ascites antibody was detected by indirect ELISA method, and the mouse anti-VZV gE monoclonal antibody was obtained after chromatography purification.

[0144] Example 2: Preparation of detection antibodies

[0145] The method for preparing monoclonal antibodies (detection antibodies) comprises the following steps:

[0146] a. Stable transfection of CHO cells to produce recombinant VZV gE protein

[0147] ① CHO cells were cultured at 2×10 5 The cells were inoculated at a density of cells / mL.

[0148] ②After cell expansion, the density was adjusted to 1×10 7 cells / mL of cell suspension, take 0.8 mL into a sterile EP tube, add 5 μg of recombinant plasmid pcDNA3.1(+)-VZV-gE (nucleotide sequence as shown in SEQ ID NO.1) and mix gently, incubate at room temperature for 15 min, and then put the mixture in an ice bath for 5 min.

[0149] ③ Set the parameters of the electroporator: voltage is 280 V, number of pulses is 3 times, each pulse time is 5 ms, pulse interval is 0.784 s, and use an electroporator cup with an inner diameter of 4 mm.

[0150] ④ Add 0.2 mL of the above mixture into the electroporation cup. Place the electroporation cup into the electroporation tank and start the electroporation program.

[0151] ⑤After the electric shock program is completed, quickly transfer the cell suspension in the electroporation cup to a 125mL conical flask containing 20mL CHO CD medium containing glutamine.

[0152] ⑥ Place the conical flask in a shaking incubator at 37°C, 8% CO2, and 125 rpm for culture.

[0153] ⑦ After culturing for 72 h, the cells were centrifuged at 1000 rpm for 5 min to remove the supernatant, and the cells were resuspended in CHO CD medium containing different concentrations of MSX (2 μM, 5 μM, and 20 μM) and the cell density and viability were monitored to maintain a cell density of approximately 1.2 × 10 6 cells / mL and the cell status was monitored every two days.

[0154] ⑧When the cell density increases to 4×10 5 cells / mL, and then 2 stable passages were performed and VIPS was used TM The monoclonal cell plating instrument implements monoclonal screening. Based on factors such as cell doubling time, aggregation rate, protein expression, etc., the most advantageous clones are selected as candidate clones for expansion culture, library construction, and further purification of the expression products.

[0155] ⑨The successful construction of the cell line was confirmed by RT-PCR and Western Blot.

[0156] ⑩ The recombinant VZV gE protein expressed in the above CHO culture system was subjected to column chromatography to obtain highly purified recombinant VZV gE protein.

[0157] b. Immunize animals to obtain serum

[0158] After recombinant VZV gE was mixed with Freund's adjuvant in a certain proportion, multiple immunizations were performed subcutaneously on the back of New Zealand rabbits. The immunization dose was 1 mg / rabbit, and each injection was 21 days apart. A total of 5 immunizations were performed, and the injection method was the same each time. Blood was collected from rabbits 21 days after the last immunization, and serum was separated by centrifugation at 2000-4000 rpm for 15 minutes. The anti-VZV gE antibody titer in the serum was detected and confirmed by ELISA.

[0159] c. Preparation of rabbit monoclonal antibody against recombinant gE protein

[0160] B lymphocytes are collected from the spleen of rabbits with high titers of VZV gE antibodies, and the B cells are separated from the lymphocyte mixture by centrifugation. The separated single B cells are cultured in a culture medium containing appropriate nutrients and growth factors. During the culture process, B cells continue to divide to form cloned cell groups, and these B cells are screened. The culture is screened using methods such as ELISA to determine the cell clones containing the desired antibodies. The selected target B cell clones are expanded and cultured, and finally the culture fluid is purified to obtain high-purity rabbit monoclonal antibodies.

[0161] Example 3: Preparation of a detection kit

[0162] The detection kit of this embodiment includes:

[0163] (1) The capture antibody and ELISA plate (96-well plate) prepared in Example 1.

[0164] (2) The detection antibody prepared in Example 2.

[0165] (3) Horseradish peroxidase-labeled antibody: HRP-labeled goat anti-rabbit IgG, HRP Conjugated, was purchased from Cosmed with the catalog number CW0103S.

[0166] (4) Preparation steps of standard products:

[0167] The recombinant gE expressed in eukaryotic cells in the CHO culture system was subjected to complex cation and molecular sieve chromatography to obtain highly purified recombinant VZV gE protein.

[0168] The recombinant VZV gE is prepared into a finished vaccine product using the same adjuvant ratio as the marketed product. Mice are immunized by injection using the same immunization method, dosage, and procedure. Serum is collected to evaluate the IgG antibody titer and the immune response capacity of several cytokines. Compared with the marketed product, it can achieve non-inferiority at a certain dose. At the same time, the purity of the purified recombinant VZV gE can reach more than 99% by high performance liquid chromatography. The recombinant VZV gE stock solution is subjected to the Lowry method for protein content determination, and the stock solution is diluted to 1.06 mg / mL according to the determination results to prepare an internal standard for standby use.

[0169] (5) Preparation of antibody diluent / sample diluent: Add an appropriate amount of BSA to 1×PBST to prepare PBST containing 1% (w / v) BSA.

[0170] (6) Single-component TMB colorimetric solution: purchased from Beijing Sizhengbai Biological Technology Co., Ltd., catalog number 4ATMB500.

[0171] (7) Preparation of stop solution: Take 10 mL of concentrated sulfuric acid (analytical grade concentrated sulfuric acid), slowly add it into 80 mL of water, and cool it for use to obtain 2N sulfuric acid as the stop solution.

[0172] (8) Preparation of washing solution: Take 50 mL of 20×PBST and add deionized water to dilute to 1000 mL to obtain 1×PBST, and mix well.

[0173] (9) Preparation of coating solution: Take 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dilute to 1 L, mix well, and adjust the pH to 9.6.

[0174] Example 4: Detection steps and methodological validation based on the detection kit

[0175] 1. Test steps based on the kit:

[0176] 1. Preparation of Materials and Reagents

[0177] (1) Coating the capture antibody ELISA plate: Dilute the capture antibody 10,000 times with the coating solution, add 100 μL / well to a 96-well ELISA plate, seal the bag, and place it horizontally at 2-8°C overnight. The next day, wash the ELISA plate three times with detergent on a plate washer and gently pat dry on clean absorbent paper.

[0178] (2) Detection antibody working solution: Dilute the detection antibody 1000 times (1:1000) with antibody diluent, mix thoroughly and set aside.

[0179] (3) Enzyme-labeled antibody working solution: Dilute the enzyme-labeled antibody 5000 times with antibody diluent, mix thoroughly and set aside.

[0180] 2. Preparation of standards and samples to be tested

[0181] (1) Standards: Prepare reference samples of different concentrations by 2-fold serial dilution using sample diluent: 15.625 ng / mL, 31.25 ng / mL, 62.5 ng / mL, 125 ng / mL, 250 ng / mL, 500 ng / mL, and 1000 ng / mL.

[0182] (2) Test samples: Use sample diluent to dilute the test samples (mouse serum) to within the linear range of the calibration curve. The final sample concentration should be multiplied by the sample dilution factor to obtain the sample concentration.

[0183] 3. Detection steps

[0184] (1) Coating: Dilute the capture antibody to 2000× and add to a 96-well ELISA plate, 100 μL / well, and incubate at 2-8°C overnight.

[0185] (2) Blocking: Add 5% (m / v) BSA-PBS for blocking and incubate at 37°C for 1 h.

[0186] (3) Sample addition: Add the sample to be tested, 100 μL / well, set up duplicate wells, seal the plate with a sealing film, and incubate at 37°C for 60 min.

[0187] (4) Washing: Discard the liquid in each well, add 350 μL of washing solution to each well, let it stand for 30 seconds, then discard the liquid in the well. Repeat 3 times. After the last wash, pat dry on absorbent paper.

[0188] (5) Add detection antibody: Add 100 μL of detection antibody working solution to each well, seal the plate with sealing film, and incubate at 37°C for 60 min.

[0189] (6) Washing: Discard the liquid in each well, add 350 μL of washing solution to each well, let it stand for 30 seconds, then discard the liquid in the well. Repeat three times. After the last wash, pat dry on absorbent paper.

[0190] (7) Adding enzyme-labeled antibody: Add 100 μL of enzyme-labeled antibody working solution to each well, seal the plate with sealing film, and incubate at 37°C for 30 min.

[0191] (8) Washing: Discard the liquid in each well, add 350 μL of washing solution to each well, let it stand for 30 seconds, then discard the liquid in the well. Repeat three times. After the last wash, pat dry on absorbent paper.

[0192] (9) Color development: Add 100 μL / well of single-component TMB color development solution to a 96-well ELISA plate, shake gently to mix, and then develop the color at room temperature in the dark for about 15 min.

[0193] (10) Stopping: Add 50 μL of stop solution to each well and mix gently.

[0194] (11) Reading: Select the wavelength of the microplate reader to be 450 nm and measure the OD of each well 450 Absorbance value.

[0195] 4. Result processing

[0196] (1) Standard curve: Calculate the OD of the reference product at each concentration 450 Mean, with standard curve concentration as horizontal axis, corresponding to OD 450 The mean is the ordinate, and the four-parameter fitting method is used for fitting and calculation to obtain the standard curve and the four-parameter fitting equation.

[0197] (2) Calculation of sample concentration: Based on the four-parameter fitting equation of the standard curve, select an OD value within the standard curve range. 450 The sample concentration is calculated from the value, and then multiplied by the sample dilution factor to get the initial sample concentration.

[0198] 2. Performance test method of the detection kit:

[0199] Including the following aspects:

[0200] 1. Exclusivity

[0201] Purpose: In the presence of other components (such as impurities, degradation products, excipients), the detection method provided in this application is used to determine the specificity of the constructed kit.

[0202] Steps: Use the purified stock solution of recombinant VZV glycoprotein E, the inactivated rabies vaccine stock solution, and the inactivated hepatitis A vaccine stock solution as antigen samples, set phosphate buffer as negative control, repeat 3 wells for each sample, and measure according to the method to observe whether each sample has an immune response.

[0203] Acceptable criteria: recombinant VZV gE stock solution is positive, and the rest of the samples are negative.

[0204] The results of the antigen content specificity test are shown in the table below.

[0205] Table 2. Antigen content specificity test

[0206] Test product name Result judgment Recombinant glycoprotein E purified stock solution Positive Inactivated rabies vaccine stock solution Negative Inactivated Hepatitis A Vaccine Stock Solution Negative Phosphate buffered saline Negative

[0207] From the above table, we can see that in the specific test for the antigen content detection of the purified stock solution of recombinant VZV glycoprotein E, only the purified stock solution of recombinant glycoprotein E was positive, while the others were negative, and the other components had no interference with the test.

[0208] 2. Accuracy

[0209] Purpose: To determine the consistency or closeness of the measured value of the constructed test method to the true value or recognized reference value.

[0210] Steps: Dilute the recombinant VZV gE standard into three different concentrations, calculate the measured concentration according to the standard curve, measure each concentration three times, and take the average value. Calculate the ratio of the measured concentration to the theoretical concentration to get the recovery rate.

[0211] Acceptable standard: The recovery rate of gE antigen at each content should be between 80% and 120%.

[0212]

[0213] The results of the antigen content accuracy test are shown in the table below.

[0214] Table 3. Antigen content accuracy test

[0215]

[0216]

[0217] By diluting the recombinant VZV gE standard at different multiples and repeating the test three times, the recovery rates of the standards at different contents were between 94.4% and 98.9%, which met the acceptable standards.

[0218] 3. Repeatability

[0219] Purpose: To determine the precision of the developed detection method within the same experiment and for the same sample within the measurement range.

[0220] Steps: Select different batches of purified recombinant VZV glycoprotein E stock solutions, and repeat the measurement three times by the same experimenter on the same day to calculate the variation dilution (CV) of each batch of samples.

[0221] Calculation formula: Coefficient of variation (CV) = standard deviation / mean value × 100%

[0222] Acceptable standard: Coefficient of variation (CV) of samples in the same batch ≤ 15%.

[0223] The results of the antigen content repeatability test are shown in the table below.

[0224] Table 4. Antigen content repeatability test

[0225]

[0226] The coefficient of variation of stock solution ① was 1.95%, and the coefficient of variation of stock solution ② was 2.26%, which met the acceptable standard.

[0227] 4. Precision

[0228] Purpose: To determine whether random variation factors have any influence on the detection method constructed in this application. Random variation factors include the variation of different time, different experimenters, and different brands of microplate readers.

[0229] Steps: Dilute the recombinant VZV gE standard to the same concentration and have two experimenters, A and B, verify it using the same batch of test kits for 3 days. Calculate the coefficient of variation (CV) of the test results of the same concentration sample at different test times, between different inspectors, and between different brands of ELISA readers.

[0230] Acceptable standard: The coefficient of variation (CV) of the test results of the same batch of samples at different test times, between different inspectors, and between different brands of ELISA readers is ≤15%.

[0231] The results of the antigen content precision test are shown in the table below.

[0232] Table 5. Antigen content precision test

[0233]

[0234] The same concentration of standard was tested three times by two experimenters at different times using two different types of microplate readers, and the coefficient of variation was 1.50%-5.34%, which met the acceptable standard.

[0235] 5. Linear range

[0236] Purpose: To prove that the test result is proportional to the concentration of the analyte in the sample within the measurement range.

[0237] Steps: The recombinant VZV gE standard (labeled amount: 1.06 mg / mL) was diluted 1000 times (1.06 μg / mL), 2000 times (0.53 μg / mL), 4000 times (0.265 μg / mL), 8000 times (0.1325 μg / mL), 16000 times (0.06625 μg / mL), and 32000 times (0.033125 μg / mL) according to the method, and the corresponding absorbance was subjected to linear correlation analysis to obtain R 2 .

[0238] Acceptable standard: R 2 ≥0.95, linearity holds.

[0239] The linear range results of antigen content are shown in the table below.

[0240] Table 6. Antigen content linearity and range test

[0241] Antigen standard content (μg / mL) 0.067-1.10 0.069-1.04 <![CDATA[R 2 Value]]> 0.9866 0.9956

[0242] The same inspector conducts multiple inspections. 2 The values ​​are 0.9866 and 0.9956 respectively, R 2 The linear range was ≥0.95. The OD value of 0.033125 μg / mL was close to zero, so it was not considered as the starting point of the linear range. The linear range was set at 0.067-1.10 μg / mL.

[0243] 6. Durability

[0244] Purpose: To prove the consistency of the test results between different color development times in this experiment.

[0245] Steps: Select different batches of purified recombinant VZV glycoprotein E stock solution, test according to the law, set the color development time to 10min, 15min, and 30min, and have the same experimenter perform the test. Calculate the coefficient of variation (CV) of each sample and observe the effect of different color development times on the test results.

[0246] Antigen content durability results are shown in the table below.

[0247] Table 7. Antigen content durability test

[0248]

[0249] The durability of antigen content was tested using different batches of stock solution, and the coefficient of variation of the test results was 6.93%-13.05%, which met the acceptable standards.

[0250] The present application is based on the designed recombinant VZV gE, prepares monoclonal antibodies, and establishes a double antibody sandwich ELISA method to determine VZV gE (including recombinant VZV gE). The present application has a simple detection method, high sensitivity, and good specificity, and provides a scientific basis for the detection of VZV gE, VEV, etc., and can be used for the quantitative detection of VZV gE content in samples at various stages of the research and development of varicella vaccine and herpes zoster vaccine. The technical features of the above-mentioned embodiments and examples can be combined in any appropriate manner. In order to make the description concise, all possible combinations of the various technical features in the above-mentioned embodiments and examples are not 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 of this specification.

[0251] 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 VZV gE-specific binding protein, characterized in that: The specific binding protein has one or more of antigen binding domain a and antigen binding domain b; in, The antigen binding domain a has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.8 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.9; The antigen binding domain b has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.10 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.

11.

2. The VZV gE-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 intrabody, a nanobody, a binding domain immunoglobulin fusion protein, a small module immune drug, a camelized antibody or an antibody containing VHH.

3. The VZV gE-specific binding protein according to any one of claims 1 to 2, characterized in that The specific binding protein includes an antibody constant region.

4. The VZV gE-specific binding protein according to claim 3, characterized in that The sequence of the antibody constant region is selected from any one of the constant regions of IgG, IgA, IgM, IgE and IgD.

5. The VZV gE-specific binding protein according to claim 3, characterized in that The antibody constant region may be derived from cattle, horses, pigs, sheep, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese or humans.

6. A nucleic acid, characterized in that The nucleic acid encodes the VZV gE-specific binding protein according to any one of claims 1 to 5.

7. A recombinant vector, characterized in that: The recombinant vector comprises the nucleic acid according to claim 6.

8. The recombinant vector according to claim 7, characterized in that The recombinant vector is a plasmid, a bacteriophage, a plant cell virus or an animal cell virus.

9. A cell, characterized in that The cell expresses the VZV gE-specific binding protein according to any one of claims 1 to 5, comprises the nucleic acid according to claim 6, or comprises the recombinant vector according to claim 7.

10. The cell according to claim 9, characterized in that The cells are CHO cells, COS cells, NSO cells, HeLa cells, BHK cells or HEK293 cells.

11. A method for constructing a cell according to any one of claims 9 to 10, characterized in that: The construction method comprises the step of introducing the nucleic acid according to claim 6 or the recombinant vector according to any one of claims 7 to 8 into the host cell to be transformed.

12. The method for constructing a cell according to claim 11, characterized in that: Methods of introduction include transfection.

13. A method for producing a specific binding protein of VZV gE according to any one of claims 1 to 5, characterized in that: The production method comprises using the cell according to any one of claims 9 to 10 to produce a specific binding protein of VZV gE.

14. Use of the VZV gE specific binding protein according to any one of claims 1 to 5 in the preparation of a detection kit.

15. A VZV gE detection kit, characterized in that: The detection kit comprises the VZV gE specific binding protein according to any one of claims 1 to 5.

16. The VZV gE detection kit according to claim 15, characterized in that: The detection kit comprises an antibody 1 having the antigen-binding domain a and an antibody 2 having the antigen-binding domain b.

17. A method for detecting VZV gE or a mutant thereof in a sample, characterized in that: The detection method comprises: The specific binding protein according to any one of claims 1 to 5 or the detection kit according to any one of claims 15 to 16 is used to detect VZV gE or a mutant thereof in a sample to be tested.

18. The method for detecting VZV gE or a mutant thereof in a sample according to claim 17, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO.

2.

19. The method for detecting VZV gE or a mutant thereof in a sample according to any one of claims 17 to 18, characterized in that: The sample to be tested includes cell fermentation broth.

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