CN112979787B

CN112979787B - Binding protein binding HBeAg and application thereof

Info

Publication number: CN112979787B
Application number: CN201911284806.7A
Authority: CN
Inventors: 崔鹏; 何志强; 孟媛; 钟冬梅; 周俊; 覃婷
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2019-12-13
Filing date: 2019-12-13
Publication date: 2022-11-08
Anticipated expiration: 2039-12-13
Also published as: CN112979787A

Abstract

The invention discloses a binding protein for binding HBeAg and application thereof, relating to the technical field of antibodies. The invention discloses a binding protein specifically binding to HBeAg, which comprises an antigen binding domain, wherein the antigen binding domain comprises at least one of the following complementarity determining regions: complementarity determining region CDR-VH1, complementarity determining region CDR-VH2 and complementarity determining region CDR-VH3. The binding protein specifically binding to HBeAg disclosed by the invention can bind to HBeAg, has better binding activity and affinity, can be used for detecting HBeAg antigen level and diagnosing HBV infection, and provides more protein choices for HBeAg detection and HBV infection diagnosis.

Description

Binding protein for binding HBeAg and application thereof

Technical Field

The invention relates to the technical field of antibodies, in particular to a binding protein for binding HBeAg and application thereof.

Background

Worldwide, about 20 million Hepatitis B Virus (HBV) infected persons are the main causes of acute and chronic liver diseases, and about 100 million people die each year from liver failure, cirrhosis and primary hepatocellular carcinoma caused by HBV infection. About 9300 million people exist in the chronic HBV infectors in China, and about 2000 million patients have chronic hepatitis B. The chronic hepatitis B still lacks effective control means and thorough solution, and the progress of molecular mechanism research on the interaction between HBV and liver cells and immune cells is relatively delayed, which is one of the important reasons for seriously affecting the development of novel therapeutic methods and drugs.

Hepatitis B virus e antigen (HBV e antigen, HBeAg) is a virological marker of HBV infection and is widely applied to clinic, and has important clinical value. HBeAg plays an important role in the chronic process after perinatal HBV infection, and the newborn of HBeAg positive mother is often infected with HBV in the perinatal period and then is infected with HBV. The conversion process of HBeAg disappearance and HBeAb appearance usually (except caused by C/C gene mutation) means that the virus replication level is reduced and the liver inflammation activity degree is weakened, so that the serological conversion of HBeAg is always used by scholars at home and abroad as one of important indexes for evaluating the curative effect of antiviral treatment.

The genome of hepatitis B virus is a small and efficient system, only 3.2kb, with only 4 major open reading frames, but is responsible for the expression of nearly ten proteins. The genome is generally divided into: the pre-S region, the CORE region (pre-c/c) responsible for the expression of two proteins: hepatitis B virus core protein and hepatitis B virus E protein. That is, HBeAg and HBcAg share the same segment of the genome. More and more studies have demonstrated that HBeAg is a protease-cleaved product of HBcAg. The molecular weight of HBcAg is 21000 daltons, and the molecular weight of HBeAg is 14000 daltons. HBeAg encodes in the pre-C region and has partial homology with the core antigen (C antigen), but is a secreted antigen due to a signal peptide at the N-terminus. The initial code AUG at nt1814 in the front C gene sequence begins, and is translated to the 3' end of the C region, the obtained expression product is the precursor of HBeAg, the signal peptide at the N end is cut by signal peptidase, the C end is degraded by cell protease to become mature HBeAg, which can be secreted into blood circulation from infected cells, thus not only leading to the chronization of HBV infection of perinatal newborn, but also leading to low immune response ability of patients to HBV and poor antiviral treatment effect when the HBV exists and presents high level.

There are many methods for detecting HBeAg, mainly a sandwich method based on antigen-antibody reaction. The method has the advantages of low cost, simple operation, suitability for large-scale screening and the like, and is the most common detection method at present. In recent years, new detection methods and techniques have been developed, including microparticle enzyme immunoassay, chemiluminescence immunoassay, and time-resolved fluorescence immunoassay, which are optimized and upgraded based on the original double antibody sandwich method. These methods are mainly based on the specific binding reaction between antigen and antibody, and in general, monoclonal antibodies with good specificity and high sensitivity are always the basis and precondition for the development of various methods and technologies.

The traditional clinical diagnosis uses mouse monoclonal antibodies which are greatly influenced by individual mice, and have unstable production, large batch difference and large mouse autoantibody purification difficulty. In addition, the use of murine monoclonal antibodies in the detection process has some disadvantages, especially when two murine monoclonal antibodies are used simultaneously in the detection reagent of the double antibody sandwich method, false positives easily appear, which cause many reasons and are difficult to analyze, for example, HAMA effect may exist in blood samples, and components capable of binding with murine antibody may exist in some pharyngeal swab or nasal swab samples. In addition, the existing anti-HBeAg antibody has low activity and poor affinity, and cannot be well applied to the detection of HBeAg. Therefore, there is a strong need in the art for antibodies that effectively and specifically bind to and can detect HBeAg.

Disclosure of Invention

The invention aims to provide a binding protein capable of specifically binding HBeAg and application thereof. The binding protein provided by the invention can be specifically bound with HBeAg, has better binding activity and affinity, can be used for detecting HBeAg and diagnosing HBV infection, and provides more choices for effective detection of HBeAg and diagnosis of HBV infection.

Noun definitions

The term "binding protein" broadly refers to all proteins/protein fragments, in particular antibodies or functional fragments of antibodies, comprising CDR regions. The term "antibody" includes polyclonal and monoclonal antibodies, and "antibody functional fragments" include antigenic compound-binding fragments of the antibodies described above, including Fab, F (ab') 2, fd, fv, scFv, diabodies, and minibody recognition units, as well as single chain derivatives of the antibodies and fragments. The type of antibody can be selected from IgG1, igG2, igG3, igG4, igA, igM, igE, igD. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional) and humanized (humanized) antibodies, as well as related synthetic isoforms (antibodies). The term "antibody" is used interchangeably with "immunoglobulin".

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are usually the most variable parts of an antibody and contain an antigen binding site. The light or heavy chain variable region (VL or VH) is composed of framework regions interrupted by three hypervariable regions, called "complementarity determining regions" or "CDRs". The extent of the framework regions and CDRs has been precisely defined, for example, in Kabat (see Sequences of Proteins of Immunological Interest), E.Kabat et al, U.S. department of Health and Human Services (U.S.. Department of Health and Human Services), (1983), and Chothia. The framework regions of the antibody, which constitute the combination of the essential light and heavy chains, serve to locate and align the CDRs, which are primarily responsible for binding to the antigen.

As used herein, "framework region" or "FR" region means the region of an antibody variable domain excluding those defined as CDRs. Each antibody variable domain framework can be further subdivided into adjacent regions (FR 1, FR2, FR3 and FR 4) separated by CDRs.

Typically, the variable domains VL/VH of the heavy and light chains are obtained by linking the CDRs and FRs numbered as follows in a combinatorial arrangement: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The term "purified" or "isolated" in relation to a polypeptide or nucleic acid, as used herein, means that the polypeptide or nucleic acid is not in its natural medium or in its natural form. Thus, the term "isolated" includes a polypeptide or nucleic acid that is removed from its original environment, e.g., from its natural environment if it is naturally occurring. For example, an isolated polypeptide is generally free of at least some proteins or other cellular components that are normally bound to or normally mixed with it or in solution. Isolated polypeptides include the naturally-produced polypeptide contained in a cell lysate, the polypeptide in purified or partially purified form, recombinant polypeptides, the polypeptide expressed or secreted by a cell, and the polypeptide in a heterologous host cell or culture. In connection with a nucleic acid, the terms "isolated" or "purified" mean that the nucleic acid is not in its natural genomic context (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).

Exemplary embodiments of the invention:

in a first aspect, embodiments of the present invention provide a binding protein that specifically binds to HBeAg, the binding protein comprising an antigen binding domain; the antigen binding domain comprises at least one of the following complementarity determining regions, or a similar complementarity determining region having at least 80% sequence identity with the sequence of at least one of the complementarity determining regions:

a complementarity determining region CDR-VH1 having an amino acid sequence of G-Y-X1-F-T-X2-Y-X3-M-N, wherein: x1 is S or T, X2 is G, D or A, X3 is D or N;

a complementarity determining region CDR-VH2 having an amino acid sequence of N-X1-D-P-Y-F-G-X2-S-X3-Y-N-X4-K, wherein: x1 is I, V or L, X2 is G, S or T, X3 is D or N, X4 is K or Q;

a complementarity determining region CDR-VH3 having the amino acid sequence X1-R-Y-X2-T-W-X3-Y-a-M, wherein: x1 is T or A; x2 is I, V or L; x3 is N or D;

a complementarity determining region CDR-VL1 having an amino acid sequence R-S-S-X1-S-X2-V-H-S-X3-G-N-T-Y-X4-E, wherein: x1 is Q or K, X2 is I, V or L, X3 is N or D, X4 is I, V or L;

a complementarity determining region CDR-VL2 having an amino acid sequence of K-V-S-X1-R-X2-S, wherein: x1 is D or N, X2 is F, V or L;

a complementarity determining region CDR-VL3 having an amino acid sequence of F-X1-G-S-H-X2-P-P, wherein: x1 is Q, H or K, and X2 is A or V.

The binding protein provided by the embodiment of the invention contains an antigen binding domain, the antigen binding domain comprises at least one of the complementarity determining regions, the amino acid sequence of the complementarity determining region is discovered and revealed for the first time, the binding protein is a novel sequence, the binding protein can be endowed with the capability of specifically binding to HBeAg antigen, has better binding activity and affinity, can be used for detecting HBeAg and diagnosing HBV infection, and provides more binding protein selections for the effective detection of HBeAg and the diagnosis of HBV infection.

In alternative embodiments, in the complementarity determining region CDR-VH1, X1 is S; in the complementarity determining region CDR-VH2, X3 is N; in the CDR-VH3, X1 is T; (ii) in said complementarity determining region CDR-VL1, X1 is Q in said complementarity determining region CDR-VL2, X1 is N; in the CDR-VL3, X2 is A.

In alternative embodiments, in the complementarity determining region CDR-VH1, X2 is G.

In alternative embodiments, in the complementarity determining region CDR-VH1, X2 is D.

In an alternative embodiment, in the complementarity determining region CDR-VH1, X2 is a.

In an alternative embodiment, in the complementarity determining region CDR-VH1, X3 is D.

In an alternative embodiment, in the complementarity determining region CDR-VH1, X3 is N.

In an alternative embodiment, in the complementarity determining region CDR-VH2, X1 is I.

In alternative embodiments, in the complementarity determining region CDR-VH2, X1 is V.

In alternative embodiments, in the complementarity determining region CDR-VH2, X1 is L.

In alternative embodiments, in the complementarity determining region CDR-VH2, X2 is G.

In an alternative embodiment, in the complementarity determining region CDR-VH2, X2 is S.

In alternative embodiments, in the complementarity determining region CDR-VH2, X2 is T.

In an alternative embodiment, in the complementarity determining region CDR-VH2, X4 is K.

In alternative embodiments, in the complementarity determining region CDR-VH2, X4 is Q.

In alternative embodiments, in the complementarity determining region CDR-VH3, X2 is I.

In alternative embodiments, in the complementarity determining region CDR-VH3, X2 is V.

In alternative embodiments, in the complementarity determining region CDR-VH3, X2 is L

In an alternative embodiment, in the complementarity determining region CDR-VH3, X3 is N.

In an alternative embodiment, in the complementarity determining region CDR-VH3, X3 is D.

In alternative embodiments, in the complementarity determining region CDR-VL1, X2 is I.

In alternative embodiments, in the complementarity determining region CDR-VL1, X2 is V.

In alternative embodiments, in the complementarity determining region CDR-VL1, X2 is L.

In alternative embodiments, in the complementarity determining region CDR-VL1, X3 is N.

In alternative embodiments, in the complementarity determining region CDR-VL1, X3 is D.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X4 is I.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X4 is V.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X4 is L.

In an alternative embodiment, in the complementarity determining region CDR-VL2, X2 is F.

In alternative embodiments, in the complementarity determining region CDR-VL2, X2 is V.

In alternative embodiments, in the complementarity determining region CDR-VL2, X2 is L.

In an alternative embodiment, in the complementarity determining region CDR-VL3, X1 is Q.

In an alternative embodiment, in the complementarity determining region CDR-VL3, X1 is H.

In alternative embodiments, in the complementarity determining region CDR-VL3, X1 is K.

In alternative embodiments, the similar complementarity determining regions have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequences of the complementarity determining regions described above.

In alternative embodiments, the antigen binding domain has a K with the HBeAg protein _D ≤3.82×10 ^-9 Affinity of mol/L;

in alternative embodiments, the antigen binding domain has a K with the HBeAg protein _D ≤3×10 ^-9 mol/L、2×10 ^-9 mol/L、1×10 ^-9 mol/L、9×10 ^-10 mol/L、8×10 ^-10 mol/L、7×10 ^-10 mol/L、6×10 ^-10 mol/L、5×10 ^-10 mol/L、4×10 ^-10 mol/L、3×10 ^-10 mol/L、2×10 ^-10 mol/L、1×10 ^-10 mol/L、9×10 ^-11 mol/L、8×10 ^-11 mol/L、7×10 ^-11 mol/L、6×10 ^-11 mol/L or 5X 10 ^-11 Affinity of mol/L;

in an alternative embodiment, the antigen binding domain has 5.52 x 10 to HBeAg protein ^-11 mol/L≤K _D ≤3.82×10 ^-9 Affinity in mol/L.

K _D The detection of (c) is carried out with reference to the method in the example of the present invention.

In an alternative embodiment, the mutation site (i.e., xn site, n =1,2,3 or 4) in each of the complementarity determining regions described above is selected from any one of the following combinations of mutations 1-57:

in alternative embodiments, in the complementarity determining region CDR-VH1, X1 is T; in the CDR-VH2, X3 is D; in the CDR-VH3, X1 is A; in the complementarity determining region CDR-VL1, X1 is K; in the complementarity determining region CDR-VL2, X1 is D; in the CDR-VL3, X2 is V.

In an alternative embodiment, the mutation site (i.e., xn site, n =1,2,3 or 4) in each of the complementarity determining regions described above is selected from any one of the following combinations of mutations 58-67:

in alternative embodiments, the binding protein includes at least 3 complementarity determining regions (e.g., 3 complementarity determining regions of a heavy chain, or3 complementarity determining regions of a light chain); alternatively, the binding protein comprises at least 6 complementarity determining regions (e.g., 3 complementarity determining regions of a heavy chain and 3 complementarity determining regions of a light chain);

in alternative embodiments, the binding protein is an intact antibody comprising a variable region and a constant region.

In alternative embodiments, the binding protein is a functional fragment of an antibody, such as any one of a nanobody, F (ab ') 2, fab', fab, fv, scFv, diabody, and antibody minimal recognition unit;

functional fragments of the above antibodies typically have the same binding specificity as the antibody from which they are derived. It will be readily understood by those skilled in the art from the disclosure of the present invention that functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reduction to cleave disulfide bonds.

Functional fragments of the above antibodies can also be obtained by recombinant genetic techniques also known to those skilled in the art or synthesized by, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems and the like.

In alternative embodiments, the binding protein comprises light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4, as shown in sequence in SEQ ID NOS: 1-4, and/or heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4, as shown in sequence in SEQ ID NOS: 5-8.

In addition, based on the disclosure of the present invention, the species source of the heavy chain or light chain framework region of the binding protein may be human, so as to constitute a humanized antibody.

In alternative embodiments, the binding protein further comprises an antibody constant region.

In alternative embodiments, the antibody constant region is selected from the constant regions of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD.

In alternative embodiments, the species of the antibody constant region is from a cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken fountains, or human.

In alternative embodiments, the antibody constant region is derived from a mouse.

In alternative embodiments, the light chain constant region sequence of the antibody constant region is set forth in SEQ ID NO. 9 and the heavy chain constant region sequence of the antibody constant region is set forth in SEQ ID NO. 10.

The sequences of SEQ ID NOS: 1-10 are shown in the following table:

in a second aspect, the present embodiments provide the use of a binding protein according to any one of the preceding embodiments in the manufacture of a reagent or kit for diagnosing HBV infection.

In a third aspect, the embodiments provide a reagent or a kit for diagnosing HBV infection, which contains the binding protein of any one of the previous embodiments.

In a fourth aspect, an embodiment of the present invention provides a method for detecting HBeAg, including: mixing a binding protein according to any one of the preceding embodiments with a sample to be tested.

In an alternative embodiment, the above method is for the purpose of non-disease diagnosis.

It should be noted that, one skilled in the art can perform qualitative or quantitative detection of HBeAg protein in the sample to be tested based on the characteristics of immune complex formed by antibody/antigen combination. The method for detecting the antigen or the antibody based on the immune complex formed by the binding of the antibody and the antigen comprises the following steps:

(1) The detection purpose is realized by a precipitation reaction, which comprises the following steps: a one-way immunodiffusion test, a two-way immunodiffusion test, an immunoturbidimetry, a convective immunoelectrophoresis, an immunoblotting method, and the like;

(2) The detection purpose is realized by marking an indicator for displaying signal intensity, and the method comprises the following steps: immunofluorescence, radioimmunoassay, enzyme-linked immunoassay, and chemiluminescent immunoassay (e.g., double antibody sandwich, indirect method, or competitive method);

the indicator may be selected appropriately according to different detection methods, including but not limited to the indicators described below:

(1) In the immunofluorescence method, the indicator may be a fluorescent dye, for example, a fluorescein-based dye (including Fluorescein Isothiocyanate (FITC), hydroxyphoton (FAM), tetrachlorofluorescein (TET), etc. or an analog thereof), a rhodamine-based dye (including red Rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or an analog thereof), a Cy-based dye (including Cy2, cy3B, cy3.5, cy5, cy5.5, cy3, etc. or an analog thereof), an Alexa-based dye (including Alexa fluor350, 405, 430, 488, 532, 546, 555, 594, 610, 33, 647, 680, 700, 750, etc. or an analog thereof), a protein-based dye (including Phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polymetaxanthin-chlorophyll protein (preCP), etc.);

(2) In radioimmunoassays, the indicator may be a radioisotope, for example: 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, 18F, and the like.

(3) In enzyme-linked immunoassays, the indicator may be an enzyme that catalyzes the development of a substrate (e.g., horseradish peroxidase, alkaline phosphatase, or glucose oxidase, etc.).

(4) In the chemiluminescent immunoassay, the indicator may be a chemiluminescent liquid such as acridinium ester, horseradish peroxidase and luminol, alkaline phosphatase and AMPPD, electrochemiluminescent agents such as ruthenium terpyridyl and tripropylamine, and the like.

Based on the disclosure of the above binding protein, those skilled in the art can easily think of using any one or a combination of several methods or other methods to achieve quantitative or qualitative detection of HBeAg in a sample to be detected, and whatever method is selected, it is within the scope of the present invention as long as the binding protein disclosed in the present invention is used to detect HBeAg.

In an alternative embodiment, the binding protein is labeled with an indicator that shows signal intensity, such that the complex in which the binding protein binds to the HBeAg protein is detected.

In a fifth aspect, embodiments of the invention provide an isolated nucleic acid encoding a binding protein according to any one of the preceding embodiments;

in alternative embodiments, the nucleic acid is DNA or RNA.

Based on the disclosure of the amino acid sequence of the binding protein, one skilled in the art can easily obtain the nucleic acid sequence encoding the binding protein according to the codon corresponding to the amino acid, and obtain various nucleic acid sequences encoding the binding protein according to the degeneracy of the codon, which are within the protection scope of the present invention as long as they encode the binding protein.

In a sixth aspect, embodiments of the invention provide a vector comprising a nucleic acid according to the previous embodiments.

Wherein the nucleic acid sequence is operably linked to at least one regulatory sequence. "operably linked" means that the nucleic acid sequence is linked to the regulatory sequence in a manner that allows expression. Regulatory sequences, including promoters, enhancers and other expression control elements, are selected to direct the expression of the protein of interest in a suitable host cell.

Herein, a vector may refer to a molecule or agent comprising a nucleic acid of the invention or a fragment thereof, capable of carrying genetic information and capable of delivering the genetic information into a cell. Typical vectors include plasmids, viruses, bacteriophages, cosmids and minichromosomes. The vector may be a cloning vector (i.e. a vector for transferring genetic information into a cell, which cell may be propagated and in which the presence or absence of the genetic information may be selected) or an expression vector (i.e. a vector comprising the necessary genetic elements to allow expression of the genetic information of the vector in a cell). Thus, a cloning vector may contain a selectable marker, as well as an origin of replication compatible with the cell type specified by the cloning vector, while an expression vector contains the regulatory elements necessary to effect expression in a specified target cell.

The nucleic acid of the invention or fragments thereof may be inserted into a suitable vector to form a cloning or expression vector carrying the nucleic acid fragment of the invention. Such novel vectors are also part of the present invention. The vector may comprise a plasmid, phage, cosmid, minichromosome, or virus, as well as naked DNA that is transiently expressed only in a particular cell. The cloning and expression vectors of the present invention are capable of autonomous replication and therefore are capable of providing high copy numbers for high level expression or high level replication purposes for subsequent cloning. The expression vector may comprise a promoter for driving expression of the nucleic acid fragment of the invention, optionally a signal peptide sequence encoding for secretion or integration of the protein expression product into a membrane, and optionally a nucleic acid sequence encoding for a terminator. When the expression vector is manipulated in a production strain or cell line, the vector, when introduced into a host cell, may or may not be integrated into the genome of the host cell. Vectors typically carry a replication site, as well as a marker sequence capable of providing phenotypic selection in transformed cells.

In a seventh aspect, embodiments of the present invention provide a host cell comprising a vector according to the previous embodiments.

The expression vectors of the invention are useful for transforming host cells. Such transformed host cells are also part of the invention and may be cultured cells or cell lines for propagation of the nucleic acid fragments and vectors of the invention, or for recombinant production of the binding proteins of the invention. The host cell of the present invention includes microorganisms such as bacteria (e.g., escherichia coli, bacillus, etc.). Host cells also include cells from multicellular organisms such as fungi, insect cells, plant cells or mammalian cells, preferably from mammals, e.g., CHO cells.

In an eighth aspect, embodiments of the invention provide a method of producing a binding protein of any one of the preceding embodiments, comprising:

the host cell of the previous embodiment is cultured, and the binding protein is isolated and purified from the culture medium or from the cultured host cell.

The production method may be, for example, transfecting a host cell with a nucleic acid vector encoding at least a portion of the binding protein, and culturing the host cell under suitable conditions such that the binding protein is expressed. The host cell may also be transfected with one or more expression vectors, which may comprise, alone or in combination, DNA encoding at least a portion of the binding protein. The bound protein may be isolated from the culture medium or cell lysate using conventional techniques for purifying proteins and peptides, including ammonium sulfate precipitation, chromatography (e.g., ion exchange, gel filtration, affinity chromatography, etc.), and/or electrophoresis.

Construction of suitable vectors containing the coding and regulatory sequences of interest can be carried out using standard ligation and restriction techniques well known in the art. The isolated plasmid, DNA sequence or synthetic oligonucleotide is cleaved, tailed and religated as desired. Any method may be used to introduce mutations into the coding sequence to produce variants of the invention, and these mutations may comprise deletions or insertions or substitutions or the like.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a reduced SDS-PAGE of the HBeAg monoclonal antibody of example 1.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit dosages herein, some methods and materials are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are well explained in the literature, e.g. "molecular cloning: laboratory Manual (Molecular Cloning: laboratory Manual), second edition (Sambrook et al, 1989); synthesis of oligonucleotides (oligo Synthesis) (m.j. gate eds., 1984); animal Cell Culture (Animal Cell Culture), ed.r.i. freshney, 1987; methods in Enzymology (Methods in Enzymology), academic Press, inc. (Academic Press, inc.), "Handbook of Experimental Immunology" ("D.M.Weir and C.C.Black well"), gene Transfer Vectors for Mammalian Cells (J.M.Miller and M.P.Calos.), "Current Protocols in Molecular Biology" (F.M.Ausubel et al., 1987), "PCR, polymerase Chain Reaction (PCR: the Polymerase Chain Reaction) (Mullis et al., 1994), and" Current Protocols in Immunology "(blood), each of which is incorporated herein by reference, cold, 1991.

Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One of ordinary skill in the relevant art will readily recognize, however, that the invention can be practiced without one or more of the specific details or with other methods. The present invention is not limited by the illustrated ordering of activities or events, as some activities may occur in different orders and/or concurrently with other activities or events. Moreover, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The restriction enzyme, prime Star DNA polymerase, in this example was purchased from Takara. The MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART ^TM The RACE cDNAamplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen corporation. Primer synthesis and gene sequencing were performed by Invitrogen corporation.

This example provides a method for preparing a recombinant antibody against HBeAg

1 construction of recombinant plasmid

(1) Primer and method for producing the same

Amplifying Heavy Chain and Light Chain 5' RACE primers:

(2) Antibody variable region gene cloning and sequencing

RNA is extracted from a hybridoma cell strain secreting an anti-HBeAg monoclonal antibody, first strand cDNA synthesis is carried out by using a SMARTERTM RACE cDNAamplification Kit and SMARTER II A Oligonucleotide and 5' -CDS primers in the Kit, and an obtained first strand cDNA product is used as a PCR amplification template. The Light Chain gene was amplified with Universal Primer AMix (UPM), nested Universal Primer A (NUP) and mIgG CKR primers, and the Heavy Chain gene was amplified with Universal Primer AMix (UPM), nested Universal Primer A (NUP) and mIgG CHR primers. The primer pair of LightChain amplifies about 0.8KB of target band, and the primer pair of Heavy Chain amplifies about 1.4KB of target band. The product was purified and recovered by agarose gel electrophoresis, and the product was inserted into pMD-18T vector after A-addition reaction with rTaq DNA polymerase, transformed into DH 5. Alpha. Competent cells, and after colonies were grown, the Heavy Chain and Light Chain genes were cloned, respectively, and sent to Invitrogen corporation for sequencing.

(3) Sequence analysis of variable region Gene of anti-HBeAg monoclonal antibody

Putting the gene sequence obtained by sequencing in an IMGT antibody database for analysis, and analyzing by using VNTI11.5 software to determine that the genes amplified by the heavy Chain primer pair and the Light Chain primer pair are correct, wherein in the gene fragment amplified by the Light Chain, the VL gene sequence is 342bp, belongs to VkII gene family, and a leader peptide sequence of 57bp is arranged in front of the VL gene sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 360bp, belongs to a VH1 gene family, and has a leader peptide sequence of 57bp in front.

(4) Construction of recombinant antibody expression plasmid

pcDNA ^TM 3.4

vector is a constructed recombinant antibody eukaryotic expression vector, and multiple cloning enzyme cutting sites such as HindIII, bamHI, ecoRI and the like are introduced into the expression vector and are named as pcDNA3.4A expression vector, which is called 3.4A expression vector for short in the following; according to the sequencing result of the antibody gene in the pMD-18T, the light chain and heavy chain gene specific primers of the anti-HBeAg antibody are designed, two ends of the primers are respectively provided with HindIII and EcoRI enzyme cutting sites and protective bases, and the primers are as follows:

a0.75 KB Light Chain gene fragment and a 1.42KB Heavy Chain gene fragment were amplified by PCR amplification. The Heavy Chain and Light Chain gene fragments are respectively subjected to double digestion by HindIII/EcoRI, the 3.4A vector is subjected to double digestion by HindIII/EcoRI, the Heavy Chain gene and the Light Chain gene are respectively connected into the 3.4A expression vector after the fragments and the vector are purified and recovered, and recombinant expression plasmids of the Heavy Chain and the Light Chain are respectively obtained.

2 Stable cell line screening

(1) Transient transfection of recombinant antibody expression plasmid into CHO cell, determination of expression plasmid activity

Plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were conditioned at 1.43X 10 ⁷ cells/ml are put into a centrifuge tube, 100 mul of plasmid is mixed with 700 mul of cells, transferred into an electric rotating cup, electrically rotated, sampled and counted on days 3, 5 and 7, and sampled and detected on day 7.

Diluting goat anti-human IgG1 mu g/ml with the coating solution to coat the microplate, wherein each well is 100 mu l and stays overnight at 4 ℃; the next day, washing with the washing solution for 2 times, and patting dry; blocking solution (20% BSA +80% PBS) was added, 120. Mu.l per well, 37 ℃,1h, patted dry; adding diluted cell supernatant at a concentration of 100 μ l/well at 37 deg.C for 60min; washing with washing solution for 5 times, and drying; adding diluted HBV positive specimen in 100. Mu.l/well at 37 deg.c for 40min; washing with the washing solution for 5 times, and drying; adding 100 mul of mouse HBeAg monoclonal antibody marked with HRP into each hole, and performing temperature control at 37 ℃ for 30min; adding a developing solution A (50 mu l/hole), adding a developing solution B (50 mu l/hole), and carrying out 10min; adding stop solution into the mixture, wherein the concentration of the stop solution is 50 mu l/hole; OD readings were taken at 450nm (reference 630 nm) on the microplate reader. The results show that the reaction OD after the cell supernatant is diluted 1000 times is still greater than 1.0, and the reaction OD of the wells without the cell supernatant is less than 0.1, which indicates that the antibody generated after the plasmid is transiently transformed has activity on HBeAg.

(2) Linearization of recombinant antibody expression plasmids

The following reagents were prepared: 50 mul Buffer, 100 mul DNA/tube, 10 mul PuvI enzyme and sterile water to 500 mul, and performing enzyme digestion in water bath at 37 ℃ overnight; extraction was performed sequentially with equal volumes of phenol/chloroform/isoamyl alcohol (lower layer) 25, followed by chloroform (aqueous phase); precipitating with 0.1 volume (water phase) of 3M sodium acetate and 2 volumes of ethanol on ice, rinsing the precipitate with 70% ethanol, removing organic solvent, re-melting with appropriate amount of sterilized water when ethanol is completely volatilized, and finally measuring the concentration.

(3) Stable transfection of recombinant antibody expression plasmid, pressurized screening of stable cell lines

Plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were conditioned at 1.43X 10 ⁷ cells/ml are put into a centrifuge tube, 100 mul of plasmid is mixed with 700 mul of cells, and the mixture is transferred into an electric rotating cup and is electrically rotated, and the next day is counted; 25 u mol/L MSX 96 hole pressure culture about 25 days.

Observing the marked clone holes with cells under a microscope, and recording the confluence degree; taking culture supernatant, and carrying out sample detection; selecting antibody concentration, transferring cell strains with high relative concentration into 24 holes, and transferring into 6 holes after 3 days; after 3 days, the seeds were kept and cultured in batches, and the cell density was adjusted to 0.5X 10 ⁶ cells/ml,2.2ml, cell density 0.3X 10 ⁶ cell/ml, 2ml for seed preservation; and (4) 7 days, carrying out batch culture supernatant sample sending detection in 6 holes, and selecting cell strains with small antibody concentration and cell diameter to transfer TPP for seed preservation and passage.

3 recombinant antibody production

(1) Cell expanding culture

After the cell recovery, the cells were first cultured in 125ml size shake flasks, inoculated with 30ml Dynamis medium at 100% volume, and placed in a shaker at a rotation speed of 120r/min, a temperature of 37 ℃ and a carbon dioxide content of 8%. Culturing for 72h, inoculating and expanding at inoculation density of 50 ten thousand cells/ml, and calculating the expanding volume according to production requirements, wherein the culture medium accounts for 100 percent. Then the culture is expanded every 72 h. When the cell quantity meets the production requirement, the seeding density is strictly controlled to be about 50 ten thousand cells/ml for production.

(2) Shake flask production and purification

Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8 percent. Feeding in a flowing mode: daily feeding was started at 72h in the flask, 3% of the initial culture volume was fed daily by HyCloneTM Cell BoostTM Feed 7a, one thousandth of the initial culture volume was fed daily by Feed 7b, and was continued up to day 12 (day 12 feeding). Glucose was supplemented with 3g/L on the sixth day. Samples were collected on day 13. Affinity purification was performed using a proteinA affinity column. Mu.g of the purified antibody (i.e., HBeAg monoclonal antibody) was subjected to reducing SDS-PAGE, and the electrophoretogram thereof was as shown in FIG. 1. Two bands were shown after reducing SDS-PAGE, 1 with 50kD of Mr (i.e., heavy chain, SEQ ID NO: 14) and 28kD of Mr (i.e., light chain, SEQ ID NO: 12).

Example 2

Detection of antibody Performance

(1) Example 1 Activity assay of antibodies and mutants thereof

Further analysis revealed that the variable region of the heavy chain of the HBeAg monoclonal antibody (WT) of example 1 is shown in SEQ ID NO 13, wherein the amino acid sequences of the complementarity determining regions of the heavy chain are as follows:

CDR-VH1：G-Y-T(X1)-F-T-D(X2)-Y-N(X3)-M-N；

CDR-VH2：N-I(X1)-D-P-Y-F-G-S(X2)-S-D(X3)-Y-N-Q(X4)-K；

CDR-VH3：A(X1)-R-Y-L(X2)-T-W-D(X3)-Y-Y-A-M；

the light chain variable region is shown as SEQ ID NO. 11, wherein the amino acid sequences of the complementarity determining regions of the light chain are as follows:

CDR-VL1：R-S-S-K(X1)-S-I(X2)-V-H-S-D(X3)-G-N-T-Y-L(X4)-E；

CDR-VL2：K-V-S-D(X1)-R-F(X2)-S；

CDR-VL3：F-Q(X1)-G-S-H-V(X2)-P-P。

based on the HBeAg monoclonal antibody of example 1, mutation is carried out on the sites related to the antibody activity in the complementarity determining region, wherein X1, X2, X3 and X4 are all mutation sites. See table 1 below.

TABLE 1 mutant sites associated with antibody Activity

And (3) detecting the binding activity:

coating sheep anti-human IgG1 microgram/ml with coating solution (PBS) for coating microporous plate at 4 deg.C and 100 microliter per hole; the next day, washing with washing solution (PBS) for 2 times, and patting dry; blocking solution (20% BSA +80% PBS) was added, 120. Mu.l per well, 37 ℃,1h, patted dry; adding the diluted monoclonal antibody in the table 1, 100 mul/hole, 37 ℃,60min; washing with washing solution for 5 times, drying, adding diluted HBV positive specimen at 37 deg.C for 40min at a volume of 100 μ l per well; washing with the washing solution for 5 times, and drying; adding another mouse HBe monoclonal antibody (purchased from market) marked with HRP, wherein each well is 100 mul, 37 ℃ and 30min; adding color development liquid A (50 μ L/well containing 2.1g/L citric acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L carbamide peroxide) and adding color development liquid B (50 μ L/well containing 1.05g/L citric acid, 0.186g/L LEDTA.2Na, 0.45g/L TMB and 0.2ml/L concentrated HCl) for 10min; stop solution (50. Mu.l/well, containing 0.75 g/EDTA-2 Na and 10.2ml/L concentrated H) was added ₂ SO ₄ ) (ii) a OD readings were taken at 450nm (reference 630 nm) on the microplate reader. The results are shown in Table 2.

TABLE 2 Activity data of antibodies and mutants thereof

Antibody concentration (ng/ml)	25	12.5	6.25	3.125	1.5625	0
							WT	1.921	1.396	0.979	0.652	0.557	0.107
Mutation 1	2.458	2.317	2.293	2.179	1.648	0.043
							Mutation 2	2.055	2.059	2.191	2.005	1.514	0.050
Mutation 3	2.072	2.047	2.007	1.937	1.534	0.064
							Mutation 4	2.082	2.131	2.084	2.072	1.427	0.077
Mutation 5	0.321	0.123	-	-	-	-
							Mutation 6	0.285	-	-	-	-	-

As can be seen from the data in Table 2, WT, as well as mutations 1-4, had better binding activity, while mutations 5 and 6 had little binding activity, indicating that the mutation pattern at the mutation sites listed in Table 1 was unpredictable.

(2) Example 1 affinity assays for antibodies and mutants thereof

Based on mutation 1, other sites were mutated, and the sequence of each mutation is shown in table 3 below.

TABLE 3 mutation sites related to antibody affinity

And (3) affinity detection:

(a) Using AMC sensors, purified antibody with PBST diluted to 10 u g/ml, recombinant HBe antigen PBST from 50 u g/ml for 2 times gradient dilution.

The operation flow is as follows: equilibration for 60s in buffer 1 (PBST), immobilized antibody for 300s in antibody solution, incubation for 180s in buffer 2 (PBST), binding for 420s in antigen solution, dissociation for 1200s in buffer 2, sensor regeneration with 10mM pH 1.69GLY solution and buffer 3, and data output. The results are shown in Table 4 (K) _D Represents the equilibrium dissociation constant, i.e., affinity; kon denotes the binding rate; kdis denotes the off-rate).

Table 4 affinity assay data

As can be seen from the data in Table 4, the KD values of the mutation 1 and the mutations 1-1 to 1-56 are lower, which indicates that the mutation 1 and the mutations 1-1 to 1-56 have better affinity, and the mutation mode of the mutation sites shown in Table 3 has no negative influence on the affinity, and the better affinity can be obtained by carrying out the mutation according to the mutation mode of the mutation sites shown in Table 3.

(b) Based on WT, mutation is carried out on other sites, and the affinity of each mutant is detected, the sequence of each mutation is shown in Table 5, and the corresponding affinity data is shown in Table 6.

TABLE 5 mutations with WT as backbone

TABLE 6 affinity detection of mutations with WT as backbone

K _D (M)

kon(1/Ms)

kdis(1/s)

K _D (M)

kon(1/Ms)

kdis(1/s)

WT

2.16E-09

1.71E+03

3.69E-06

WT 1-5

1.54E-09

2.47E+03

3.80E-06

WT 1-1

1.84E-09

1.85E+03

3.40E-06

WT 1-6

2.36E-09

1.63E+03

3.85E-06

WT 1-2

2.36E-09

1.46E+03

3.44E-06

WT 1-7

1.39E-09

2.60E+03

3.61E-06

WT 1-3

3.82E-09

1.03E+03

3.93E-06

WT 1-8

1.26E-09

1.84E+03

2.31E-06

WT 1-4

2.71E-09

1.07E+03

2.90E-06

WT1-9

1.67E-09

2.30E+03

3.84E-06

As can be seen from the data in Table 6, WT, WT 1-WT 1-9 also had a lower K _D The results show that the affinity of WT, WT 1-WT 1-9 was also good, and that the mutation pattern at the mutation site listed in Table 5 did not affect the affinity much, and that the mutation pattern was able to obtain a better affinity.

(3) Detection of naked antibody stability

Placing the antibody in a temperature range of 4 ℃ (refrigerator), -80 ℃ (refrigerator) and 37 ℃ (thermostat) for 21 days, taking samples in 7 days, 14 days and 21 days for state observation, and performing activity detection on the samples in 21 days, wherein the result shows that under three examination conditions, no obvious protein state change is seen in 21 days of placing the antibody, and the activity does not decrease along with the rise of the examination temperature, which indicates that the self-produced antibody is stable. The following table shows the OD results of 21-day enzyme immunity activity tests of the mutant 1 antibody.

TABLE 7

Antibody concentration (ng/ml)	6.25	1.5625	0
				Samples at 4 ℃ for 21 days	2.353	1.643	0.052
21 days samples at-80 deg.C	2.467	1.624	0.044
				21 day samples at 37 deg.C	2.390	1.699	0.065

From table 7, it is seen that the antibody of the embodiment of the present invention can still detect the antigen after being stored for 21 days at different temperatures, which indicates that the antibody provided by the embodiment of the present invention has better stability, and each mutant does not affect the stability of the antibody.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Dongguan City of Pengzhi Biotech Co., ltd

<120> binding protein binding to HBeAg and application thereof

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Claims

1. A binding protein that specifically binds HBeAg, wherein the binding protein comprises an antigen binding domain; the antigen binding domain includes the following 6 complementarity determining regions:

a complementarity determining region CDR-VH1 having an amino acid sequence of G-Y-X1-F-T-X2-Y-X3-M-N, wherein: x1 is S;

a complementarity determining region CDR-VH2 having an amino acid sequence of N-X1-D-P-Y-F-G-X2-S-X3-Y-N-X4-K, wherein: x3 is N;

a complementarity determining region CDR-VH3 having the amino acid sequence X1-R-Y-X2-T-W-X3-Y-a-M, wherein: x1 is T;

a complementarity determining region CDR-VL1 having an amino acid sequence R-S-S-X1-S-X2-V-H-S-X3-G-N-T-Y-X4-E, wherein: x1 is Q;

a complementarity determining region CDR-VL2 having an amino acid sequence of K-V-S-X1-R-X2-S, wherein: x1 is N;

a complementarity determining region CDR-VL3 having an amino acid sequence of F-X1-G-S-H-X2-P-P, wherein: x2 is A;

the mutation site of each complementarity determining region of the antigen binding domain is selected from any one of the following mutation combinations 1-57:

combination of mutations CDR-VH1 X2/X3 CDR-VH2 X1/X2/X4 CDR-VH3 X2/X3 CDR-VL1 X2/X3/X4 CDR-VL2 X2 CDR-VL3 X1 Mutant combination 1 D/N I/S/Q L/D I/D/L F Q Combination of mutations 2 G/N V/G/K L/N I/N/L L H Combination of mutations 3 A/N L/T/Q V/D VD/L V K Combination of mutations 4 D/D I/G/K V/N V/N/L L H Combination of mutations 5 G/D V/T/Q I/D L/D/L V K Combination of mutations 6 A/D L/S/K I/N L/N/L F Q Mutant combination 7 D/D I/T/Q V/N L/N/V V K Combination of mutations 8 D/N V/S/K I/D L/D/V F Q Combination of mutations 9 G/N L/G/Q L/N V/N/V L H Combination of mutations 10 G/D I/S/K V/D V/D/V V K Combination of mutations 11 A/D V/G/Q I/N I/N/V L H Combination of mutations 12 A/N L/T/K L/D I/D/V F Q Mutant combination 13 G/N I/G/Q I/N V/D/I L H Combination of mutations 14 A/D V/T/K I/D V/N/I F Q Combination of mutations 15 D/N L/S/Q V/N I/D/I V K Combination of mutations 16 G/D I/T/K V/D I/N/I F Q Mutant combinations 17 A/N V/S/Q L/N L/D/I V K Mutant combinations 18 D/D L/G/K L/D L/N/I L H Combination of mutations 19 A/D V/T/K V/N L/N/V V K Combination of mutations 20 D/N I/S/Q V/D L/D/V F Q Combination of mutations 21 G/D L/T/K I/N I/N/L L H Mutant combination 22 A/N I/G/Q I/D I/D/L F Q Mutant combination 23 D/D V/G/K L/N V/N/I L H Mutant combinations 24 G/N L/G/Q L/D V/D/I V K Mutant combinations 25 D/D I/S/K V/N L/D/L L H Mutant combinations 26 D/N V/T/Q I/D L/N/L V K Mutant combinations 27 G/N L/T/Q L/N V/N/V F Q Mutant combinations 28 G/D I/G/K V/D V/D/V V H Mutant combinations 29 A/D V/S/K I/N I/D/I L Q Combination of mutations 30 A/N L/S/K L/D I/N/I F K Combination of mutations 31 D/N I/T/K I/N VD/L L Q Mutant combinations 32 G/N V/G/Q I/D V/N/L F H Mutant combination 33 A/N L/S/Q V/N I/N/V V K Mutant combinations 34 D/D I/T/Q V/D I/D/V F H Combination of mutations 35 G/D L/G/K L/N L/D/I V Q Combination of mutations 36 A/D V/T/K L/D L/N/I L K Mutant combinations 37 G/N I/S/Q L/D L/N/V F Q Combination of mutations 38 A/D I/G/K L/N V/D/I L H Mutant combinations 39 D/N I/T/Q V/D I/D/L V K Combination of mutations 40 G/D I/S/K V/N V/N/I L H Mutant combination 41 A/N I/G/Q I/D I/N/L V K Combination of mutations 42 D/D I/T/K I/N L/D/V F Q Combination of mutations 43 A/D V/S/Q V/N I/D/I V K Mutant combinations 44 D/N V/G/K V/D V/N/V F Q Combination of mutations 45 G/D V/T/Q I/N VD/L L H Mutant combinations 46 A/N V/S/K I/D I/N/I V K Mutant combinations 47 D/D V/G/Q L/N V/N/L F H Mutant combinations 48 G/N V/T/K L/D I/N/V L Q Mutant combinations 49 D/D L/S/Q V/N L/D/L F H Mutant combinations 50 D/N L/G/K I/D L/D/I L Q Combination of mutations 51 G/N L/T/Q L/N V/D/V V K Mutant combinations 52 G/D L/S/K V/D I/D/V L Q Mutant combination 53 A/D L/G/Q I/N L/N/L V K Mutant combinations 54 A/N L/T/K L/D L/N/I F H Mutant combinations 55 G/N V/T/K V/N L/N/V V K Mutant combinations 56 A/D I/S/Q V/D V/D/I L Q Mutant combinations 57 D/N L/T/K I/N I/D/L F H

。

2. A binding protein that specifically binds HBeAg, wherein the binding protein comprises an antigen binding domain; the antigen binding domain includes the following 6 complementarity determining regions:

the amino acid sequence of the complementarity determining region CDR-VH1 is G-Y-X1-F-T-X2-Y-X3-M-N;

a complementarity determining region CDR-VH2, the amino acid sequence of which is N-X1-D-P-Y-F-G-X2-S-X3-Y-N-X4-K;

a complementarity determining region CDR-VH3, the amino acid sequence of which is X1-R-Y-X2-T-W-X3-Y-Y-A-M;

a complementarity determining region CDR-VL1, the amino acid sequence of which is R-S-S-X1-S-X2-V-H-S-X3-G-N-T-Y-X4-E;

a complementary determining region CDR-VL2, the amino acid sequence of which is K-V-S-X1-R-X2-S;

a complementarity determining region CDR-VL3, the amino acid sequence of which is F-X1-G-S-H-X2-P-P;

in the CDR-VH1, X1 is T;

in the CDR-VH2, X3 is D;

in the CDR-VH3, X1 is A;

in the complementarity determining region CDR-VL1, X1 is K;

in the complementarity determining region CDR-VL2, X1 is D;

in the complementarity determining region CDR-VL3, X2 is V;

the mutation site of each complementarity determining region is selected from any one of the following combinations of mutations 58-67:

combination of mutations CDR-VH1 X2/X3 CDR-VH2 X1/X2/X4 CDR-VH3 X2/X3 CDR-VL1 X2/X3/X4 CDR-VL2 X2 CDR-VL3 X1 Mutant combinations 58 D/N I/S/Q L/D I/D/L F Q Mutant combinations 59 A/N L/T/K L/D V/D/L F K Mutant combinations 60 A/D V/T/Q L/D V/D/V L K Mutant combinations 61 D/D I/T/Q I/N L/N/I L K Mutant combinations 62 G/N I/G/Q I/D I/N/I F H Mutant combinations 63 G/D L/T/Q V/D V/N/V F H Mutant combinations 64 A/N V/T/K V/N L/N/I V H Mutant combinations 65 G/N I/G/K I/N V/D/L L Q Mutant combinations 66 A/N L/G/K V/D L/D/L F Q Mutant combinations 67 G/D V/T/K I/D L/D/V F H

。

3. The binding protein according to any one of claims 1-2, wherein said binding protein is an antibody or a functional fragment thereof.

4. The binding protein of claim 3, wherein said binding protein is selected from any one of F (ab ') 2, fab', fab, fv, scFv, and diabody.

5. The binding protein of claim 3, wherein the binding protein comprises light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 having the sequences shown in SEQ ID NOS 1-4 in sequence, and/or heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 having the sequences shown in SEQ ID NOS 5-8 in sequence.

6. The binding protein according to claim 3, wherein said binding protein further comprises an antibody constant region.

7. The binding protein of claim 6, wherein the antibody constant region is selected from the constant regions of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD.

8. The binding protein of claim 6, wherein the species of said antibody constant region is from a bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human species.

9. The binding protein according to claim 8, wherein said bovine is selected from the group consisting of dairy cattle; or, the chicken is selected from turkey or turkey.

10. The binding protein of claim 6, wherein said antibody constant region is derived from a mouse.

11. The binding protein of claim 10, wherein said antibody constant region light chain constant region sequence is set forth in SEQ ID No. 9 and said antibody constant region heavy chain constant region sequence is set forth in SEQ ID No. 10.

12. A reagent or kit comprising a binding protein according to any one of claims 1 to 11.

13. Use of a binding protein according to any one of claims 1-11 for the preparation of a kit for the detection of HbeAg in a test sample.

14. The use according to claim 13, wherein the kit is for: mixing the binding protein of any one of claims 1-11 with a sample to be tested;

the detection of HBeAg is realized by precipitation reaction or by marking an indicator showing signal intensity.

15. The use according to claim 14, wherein the method for detecting HBeAg by precipitation reaction is selected from any one or more of the following methods: one-way immunodiffusion assay, two-way immunodiffusion assay, immunoturbidimetry, immunoelectrophoresis, and immunoblotting.

16. Use according to claim 15, wherein said immunoelectrophoresis comprises convection immunoelectrophoresis.

17. The use according to claim 14, wherein the method for detecting HBeAg by marking an indicator showing signal intensity is selected from any one or more of the following methods: immunofluorescence, radioimmunoassay, chemiluminescent immunoassay, and enzyme-linked immunoassay.

18. The use according to claim 17, wherein the indicator is selected from any one of a fluorescent dye, a radioisotope, a chemiluminescent solution and an enzyme that catalyzes the color development of a substrate.

19. A vector comprising a nucleic acid encoding the binding protein according to any one of claims 1 to 11.

20. A host cell comprising the vector of claim 19.

21. A method of producing the binding protein of any one of claims 1 to 11, comprising:

culturing the host cell of claim 20, and isolating and purifying the binding protein from the culture medium or from the cultured host cell.