CN115677853A - anti-HBeAg antibody or antigen binding fragment thereof and application thereof - Google Patents

anti-HBeAg antibody or antigen binding fragment thereof and application thereof Download PDF

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CN115677853A
CN115677853A CN202110861238.3A CN202110861238A CN115677853A CN 115677853 A CN115677853 A CN 115677853A CN 202110861238 A CN202110861238 A CN 202110861238A CN 115677853 A CN115677853 A CN 115677853A
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孟媛
钟冬梅
周俊
黄玉玲
熊俊文
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Dongguan Pengzhi Biotechnology Co Ltd
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Abstract

The invention belongs to the technical field of antibodies. In particular to an anti-HBeAg antibody or an antigen binding fragment thereof and application thereof. The anti-HBeAg antibody provided by the invention can be specifically combined with HBeAg, has high affinity to HBeAg, has low cost and high stability, and can be widely applied to detection/diagnosis of HBV infection and preparation of a reagent/kit for detection/diagnosis of HBV infection.

Description

anti-HBeAg antibody or antigen binding fragment thereof and application thereof
Technical Field
The invention belongs to the technical field of antibodies. More particularly, it relates to anti-HBeAg antibodies or antigen-binding fragments thereof and uses thereof.
Background
Hepatitis B Virus (HBV) infection is one of the major causes of liver disease worldwide, and more than 20 million people are currently infected with hepatitis b virus. This malaria is transmitted to a large extent by exposure to viral-containing body fluids, including unprotected sexual contact, blood transfusion, reuse of contaminated needles and syringes, transmission from mother to baby during childbirth, and the like. Hepatitis B is a serious infectious disease seriously harming human health, and China is a big country infected by hepatitis B. Chronic hepatitis B is a chronic inflammatory disease of liver caused by persistent infection of hepatitis B virus, an effective control means and a thorough solution are still lacked for chronic hepatitis B, and the research progress of molecular mechanisms of interaction between HBV and liver cells and immune cells is relatively lagged, so that the chronic hepatitis B is one of important reasons which seriously affect the research and development of novel treatment methods and medicines.
Hepatitis B e antigen (HBeAg) is a virological marker of HBV infection and is widely applied to clinic, and has important clinical value. HBeAg is not an essential structural component of virions, and it does not appear to be involved in the viral replication cycle. HBeAg plays an important role in the chronic process after infection of HBV in perinatal period, and the newborn of HBeAg positive mother is often infected with HBV in perinatal period, so that chronic HBV infection occurs. The conversion process by which HBeAg disappears and HBeAb appears, typically (except due to pre-C/C gene variation) means a reduction in the level of viral replication and a reduction in the degree of liver inflammatory activity; therefore, scholars at home and abroad always convert HBeAg serology into HBeAg serology which is one of important indexes for evaluating the curative effect of antiviral therapy.
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 commonly used detection method at present. In recent years, new detection methods and techniques are developed, including microparticle enzyme immunoassay, chemiluminescence immunoassay, time-resolved fluorescence immunoassay, and the like, which are optimized and upgraded based on the original double antibody sandwich method. Most of these methods are based on antigen-antibody specific binding reactions, 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.
At present, the HBeAg detection antibody has few sources, and the properties of affinity, sensitivity, specificity and the like have defects. Therefore, there is a need for antibodies for detecting HBeAg with better performance and better effect.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defect and the defect of poor performance of the existing antibody for detecting HBeAg, and provides an anti-HBeAg antibody or an antigen binding fragment thereof and application thereof. The anti-HBeAg antibody of the invention has high affinity, high binding activity and high stability to HBeAg, and can be widely applied to the detection/diagnosis of HBV infection.
It is an object of the present invention to provide an anti-HBeAg antibody or antigen binding fragment thereof, said antibody comprising the following CDRs:
HCDR1 with an amino acid sequence shown as SEQ ID NO.1, HCDR2 with an amino acid sequence shown as SEQ ID NO.2 and HCDR3 with an amino acid sequence shown as SEQ ID NO. 3; the amino acid sequence is LCDR1 shown in SEQ ID NO.4, the amino acid sequence is LCDR2 shown in SEQ ID NO.5, and the amino acid sequence is LCDR3 shown in SEQ ID NO. 6.
It is another object of the invention to provide nucleic acids, vectors or cells related to the anti-HBeAg antibodies or antigen binding fragments thereof.
The invention also provides the application of the anti-HBeAg antibody or the antigen binding fragment thereof, and related nucleic acid, vector or cell thereof in preparing a reagent/kit for detecting/diagnosing HBV infection.
The invention also provides a reagent/kit for detecting/diagnosing HBV infection, which contains the antibody or antigen-binding fragment thereof, the nucleic acid, the vector or the cell.
The invention also provides a method for detecting HBeAg, which comprises the step of contacting the antibody or the antigen binding fragment thereof with a sample to be detected.
Drawings
FIG. 1 shows the result of reducing SDS-PAGE of the 11B5RMb1 antibody.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
The present invention relates to an anti-HBeAg antibody or antigen binding fragment thereof, said antibody comprising the following CDRs:
HCDR1 with an amino acid sequence shown as SEQ ID NO.1, HCDR2 with an amino acid sequence shown as SEQ ID NO.2 and HCDR3 with an amino acid sequence shown as SEQ ID NO. 3; the amino acid sequence is LCDR1 shown in SEQ ID NO.4, the amino acid sequence is LCDR2 shown in SEQ ID NO.5, and the amino acid sequence is LCDR3 shown in SEQ ID NO. 6.
In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, so long as they exhibit the desired biological activity. The term "antigen-binding fragment" is a substance that comprises a portion or all of an antibody CDR that lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to an antigen and can compete with other antigen binding molecules (including whole antibodies) for binding to a given epitope. Such fragments are selected from Fab (consisting of a complete light chain and Fd), fv (consisting of VH and VL), scFv (single chain antibody, connected by a linker peptide between VH and VL) or single domain antibody (consisting of VH only). Such fragments may be produced by recombinant nucleic acid techniques, or may be produced by enzymatic or chemical cleavage of antigen binding molecules, including intact antibodies. In the specific embodiment of the invention, the 11B5RMb1 antibody can specifically bind to HBeAg and has good binding activity and affinity for HBeAg.
In the present invention, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and to regions comprising one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment thereof for the antigen or epitope that it recognizes. The heavy chain complementarity determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR1; the light chain complementarity determining regions are denoted by LCDR, which includes LCDR1, LCDR2, and LCDR1. CDR labeling methods commonly used in the art include: the Kabat numbering scheme, the Chothia and Lesk numbering scheme, and the 1997 New standardized numbering system introduced by Lefranc et al for all protein sequences of the immunoglobulin superfamily. Kabat et al was the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of the Kabat database, and the Kabat numbering scheme is generally considered to be a widely adopted standard for numbering antibody residues. The invention adopts Kabat annotation standard to mark CDR area, but other methods to mark CDR area also belong to the protection scope of the invention. In a particular embodiment of the invention, the CDRs refer to the hypervariable regions of the heavy and light chains of the 11B5RMb1 antibody.
In some embodiments, the antibody further comprises at least one of a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 8.
In some embodiments, the antibody further comprises a heavy chain constant region and a light chain constant region; the heavy chain constant region is any one or more of IgG1, igG2, igG3, igG4, igA, igD, igE and IgM, and the light chain constant region is a kappa chain or a lambda chain.
In some embodiments, the species of the heavy and light chain constant regions are from cattle, horses, cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, geese, turkeys, banisters, or humans.
In some embodiments, the amino acid sequence of the heavy chain of the antibody is set forth in SEQ ID NO. 9, and the amino acid sequence of the light chain of the antibody is set forth in SEQ ID NO. 10.
In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, fab ', F (ab') 2 scFv, fv, fd, single chain antibody, diabody or domain antibody.
The invention also relates to nucleic acids encoding the antibodies or antigen-binding fragments thereof.
The nucleic acid is typically RNA or DNA, and the nucleic acid molecule may be single-stranded or double-stranded. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. DNA nucleic acid is used when it is ligated to a vector.
The invention also relates to vectors containing said nucleic acids.
The invention also relates to a cell containing said nucleic acid or said vector.
In addition, the use of the antibody or antigen-binding fragment thereof, the nucleic acid, the vector or the cell in the preparation of a reagent/kit for detecting/diagnosing HBV infection is within the scope of the present invention.
The present invention also relates to a reagent/kit for detecting/diagnosing HBV infection, comprising said antibody or antigen-binding fragment thereof, said nucleic acid, said vector or said cell.
In some embodiments, the kit is an immunochromatographic assay kit, an enzyme immunoassay kit, a chemiluminescent kit, or an immunoturbidimetric assay kit.
In some embodiments, the kit may include a test strip or card onto which the liquid sample from the subject is placed, or an ELISA assay plate having a well in which a liquid sample from a single subject may be placed. In some embodiments, the kit can include a testing device configured for use in a flow cytometer, bioanalyzer, biosensor.
In some embodiments, the anti-HBeAg antibody contained in the kit may be in the form of a liquid solution, attached to a solid support, or as a dry powder. When the anti-HBeAg antibody is a liquid solution, the liquid solution may be an aqueous solution. When the anti-HBeAg antibody is in a form attached to a solid support, the preferred solid support may be a chromatographic medium such as a membrane, test strip, plastic bead or plate, or a microscope slide. When the anti-HBeAg antibody is a dry powder, the powder can be reconstituted by the addition of a suitable solvent.
The invention also relates to a method for detecting HBeAg, and the antibody or the antigen-binding fragment thereof is contacted with a sample to be detected.
The above method is intended for diagnosis of non-diseases. The skilled in the art can use the anti-HBeAg antibody of the invention to perform qualitative or quantitative detection on HBeAg in a sample to be detected 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 countercurrent immunoelectrophoresis, an immunoblotting, 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, chemiluminescence immunoassay, and enzyme-linked immunoassay (e.g., double antibody sandwich, indirect, or competitive).
The invention has the following beneficial effects:
the anti-HBeAg antibody provided by the invention can be specifically combined with HBeAg, has high affinity to HBeAg, has low cost and high stability, and can be widely applied to detection/diagnosis of HBV infection and preparation of a reagent/kit for detection/diagnosis of HBV infection.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
In the following examples, restriction enzymes, prime Star DNA polymerase, were purchased from Takara. The MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TM RACE cDNA Amplification 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 done by Invitrogen.
EXAMPLE 1 preparation of anti-HBeAg antibody
1. Expression plasmid construction
(1) Gene preparation of anti-HBeAg antibody (11B 5RMb1 antibody)
mRNA is extracted from a hybridoma cell strain secreting 11B5RMb1 antibody, a DNA product is obtained by an RT-PCR method, the product is added with A by rTaq DNA polymerase for reaction and then inserted into a pMD-18T vector, the product is transformed into DH5 alpha competent cells, after colonies grow out, 4 clones of Heavy Chain (Heavy Chain) and Light Chain (Light Chain) genes are respectively cloned and sent to a gene sequencing company for sequencing.
(2) Sequence analysis of 11B5RMb1 antibody variable region Gene
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 segment amplified by the Light Chain, the gene sequence of a Light Chain variable region (VL) is 339bp, belongs to a VkII gene family, and a leader peptide sequence of 57bp is arranged in front of the gene segment; in the gene fragment amplified by the Heavy Chain primer pair, the gene sequence of a Heavy Chain variable region (VH) is 357bp, belongs to a VH1 gene family, and a leader peptide sequence of 57bp is arranged in front of the VH1 gene family.
(3) Construction of recombinant antibody expression plasmid
pcDNA TM 3.4
Figure BDA0003185827220000061
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 named as pcDNA3.4A expression vector, and the vector is called as 3.4A expression vector for short in the following; according to the sequencing result of the antibody variable region gene in the pMD-18T vector, VL and VH gene specific primers of the 11B5RMb1 antibody are designed, the two ends of the primers are respectively provided with HindIII and EcoRI enzyme cutting sites and protective bases, and a 0.72KB Light Chain gene fragment and a 1.42KB Heavy Chain gene fragment are amplified by a PCR amplification method.
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 selection
(1) Transient transfection of recombinant antibody expression plasmid into CHO cell, determination of expression plasmid activity
Plasmid was diluted to 40. Mu.g/100. Mu.L with ultrapure water and CHO cells were conditioned at 1.43X 10 7 cells/mL in a centrifuge tube, 100. Mu.L plasmid and 700. Mu.L cells mixed, transferred to an electric rotor,and (4) performing electrotransfer, sampling and counting on 3 th, 5 th and 7 th days, and collecting and detecting on 7 th day.
Coating liquid (main component is NaHCO) 3 ) Dilute HBeAg-5# (available from fapeng biosome) to 3 μ g/mL, 100 μ L per well, overnight at 4 ℃; the next day, the washing solution (Na as the main component) 2 HPO 4 NaCl) for 2 times, patting dry; add blocking solution (20% BSA +80% PBS), 120 μ L per well, 37 deg.C, 1h, pat dry; adding diluted cell supernatant at 100 μ L/well, 37 deg.C for 30min (partial supernatant for 1 h); washing with washing solution for 5 times, and drying; adding goat anti-mouse IgG-HRP (goat anti-mouse IgG-HRP) with the concentration of 100 mu L per well at 37 ℃ for 30min; washing with washing solution for 5 times, and drying; adding a developing solution A (50 muL/hole, the main components of the developing solution A are citric acid, sodium acetate, acetanilide and carbamide peroxide), adding a developing solution B (50 muL/hole, the main components of the developing solution B are citric acid, EDTA & 2Na, TMB and concentrated HCl), and carrying out 10min; adding stop solution (the main components of the stop solution are EDTA-2 Na and concentrated H) 2 SO 4 ) 50 μ L/well; 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 mu L of Buffer, 100 mu g/tube of DNA, 10 mu L of PvuI enzyme and sterile water to 500 mu L, and carrying out enzyme digestion in water bath at 37 ℃ overnight; extraction was performed sequentially with equal volumes of phenol/chloroform/isoamyl alcohol (lower layer) 25; 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 with ultrapure water to 40. Mu.g/100. Mu.L and CHO cells were conditioned at 1.43X 10 7 Putting cells/mL into a centrifuge tube, mixing 100 mu L of plasmid and 700 mu L of cells, transferring into an electric rotating cup, electrically rotating, and counting the next day; 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 cell strains with high antibody concentration and relative concentration, transferring the cell strains into 24 holes, and transferring the cell strains into 6 holes after 3 days; after 3 days, the seeds are preserved and cultured in batches, and the cell density is adjusted to be 0.5 multiplied by 10 6 cells/mL,2.2mL, at a cell density of 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. 11B5RMb1 antibody preparation
(1) Cell expanding culture
After the cells were recovered, they were cultured in 125 mL-sized shake flasks, inoculated with 30mL Dynamis medium at a medium volume of 100%, and placed in a shaker at a rotation speed of 120r/min and a temperature of 37 ℃ with 8% carbon dioxide. Culturing for 72h, inoculating and expanding at an inoculation density of 50 kilocells/mL, the expanding volume being calculated according to the production requirements, the medium being 100% Dynamis medium. Then carrying out propagation every 72 h. When the cell amount meets the production requirement, the production is carried out by strictly controlling the inoculation density to be about 50 ten thousand cells/mL.
(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. And (3) carrying out affinity purification by using a proteinA affinity chromatography column to obtain the 11B5RMb1 antibody. 6.6. Mu.g of the 11B5RMb1 antibody was subjected to reducing SDS-PAGE.
The results of reducing SDS-PAGE of the 11B5RMb1 antibody are shown in FIG. 1, which shows two bands, one at 50kD (heavy chain) and the other at 28kD (light chain).
The amino acid sequence of HCDR1 of the 11B5RMb1 antibody is shown as SEQ ID NO.1, the amino acid sequence of HCDR2 is shown as SEQ ID NO.2, and the amino acid sequence of HCDR3 is shown as SEQ ID NO. 3; the amino acid sequence of LCDR1 is shown as SEQ ID NO.4, the amino acid sequence of LCDR2 is shown as SEQ ID NO.5, and the amino acid sequence of LCDR3 is shown as SEQ ID NO. 6; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8; the amino acid sequence of the heavy chain is shown as SEQ ID NO. 9, and the amino acid sequence of the light chain is shown as SEQ ID NO. 10.
Example 2 affinity assay for anti-HBeAg antibody
Using AMC sensors, the 11B5RMb1 antibody prepared in example 1 and a commercially available control antibody were diluted to 10. Mu.g/mL with PBST, and HBeAg-5# (available from Fipeng organisms) was diluted with PBST in a gradient;
the operation flow is as follows: buffer 1 (PBST, main ingredient Na) 2 HPO 4 + NaCl + TW-20) for 60s, immobilized antibody in antibody solution for 300s, buffer 2 (PBST, main component Na) 2 HPO 4 + NaCl + TW-20) for 180s, bound 420s in antigen solution, dissociated 1200s in buffer 2, incubated with 10mM pH 1.69GLY solution and buffer 3 (PBST, na as the main component) 2 HPO 4 + NaCl + TW-20) to output data.
The results of the affinity analysis of the 11B5RMb1 antibody are shown in table 1, and show that the 11B5RMb1 antibody has significantly higher affinity for HBeAg than the control antibody.
TABLE 1
Sample name KD(M) kon(1/Ms) kdis(1/s)
Control antibodies 9.48E-08 8.68E+03 8.23E-04
11B5RMb1 antibody 9.51E-09 8.91E+04 8.47E-04
Note: in table 1, KD represents the equilibrium dissociation constant, i.e., affinity; kon denotes the binding rate; kdis denotes the off-rate. The lower the KD value, the higher the affinity.
Example 3 Activity identification of anti-HBeAg antibody
Coating liquid (main component is NaHCO) 3 ) Diluting HBeAg-5# (available from Fipeng organisms) to 3 μ g/mL, 100 μ L per well, overnight at 4 ℃; the next day, the washing solution (Na as the main component) 2 HPO 4 + NaCl) for 2 times, patting dry; add blocking solution (20% BSA +80% PBS), 120 μ L per well, 37 deg.C, 1h, pat dry; adding diluted 11B5RMb1 antibody prepared in example 1 and control antibody at 100. Mu.L/well at 37 deg.C for 30min; washing with washing solution for 5 times, and drying; adding goat anti-mouse IgG-HRP, 100uL per well, 37 ℃,30min; washing with washing solution for 5 times, and drying; adding a developing solution A (50 uL/hole), adding a developing solution B (50 uL/hole), and carrying out 10min; adding stop solution at 50 uL/hole; OD readings were taken at 450nm (reference 630 nm) on the microplate reader.
The activity identification results of the 11B5RMb1 antibody are shown in table 2, and the results show that the activity of the 11B5RMb1 antibody on HBeAg is significantly higher than that of the control antibody.
TABLE 2
Sample concentration (ng/mL) 62.5 31.25 15.625 7.813 3.906 0
Control antibodies 1.793 0.726 0.445 0.189 0.118 0.070
11B5RMb1 antibody 1.823 0.998 0.653 0.267 0.135 0.058
Note: in Table 2, the higher the OD value, the better the activity of the antibody against HBeAg.
Example 4 stability assessment of anti-HBeAg antibody
The 11B5RMb1 antibody prepared in example 1 was allowed to stand at 4 ℃ C (refrigerator), -80 ℃ C (refrigerator), and 37 ℃ C (incubator) for 21 days, and samples for 7 days, 14 days, and 21 days were observed for their states, and the activity of the samples for 21 days was measured (the activity of the samples was examined using the OD results obtained in the enzyme immunoassay).
The results of the 11B5RMb1 antibody stability test are shown in Table 3, and the results show that no obvious protein state change is seen after the antibody is placed for 21 days under three examination conditions, and the activity does not decrease with the increase of the examination temperature, which indicates that the 11B5RMb1 antibody prepared by the invention has high stability.
TABLE 3
Sample concentration (ng/mL) 31.25 15.625 0
Samples at 4 ℃ for 21 days 1.829 0.858 0.094
21 day samples at-80 deg.C 1.835 0.831 0.083
21-day samples at 37 deg.C 1.833 0.828 0.067
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.
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Val Leu His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Leu
35 40 45
Gly Tyr Leu Leu Pro Tyr Asn Asp Gly Thr Asn Tyr Thr Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Pro Leu Ser Tyr Gly Asp Tyr Leu Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr
115 120 125
Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu
130 135 140
Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp
145 150 155 160
Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser
180 185 190
Thr Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala Ser
195 200 205
Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys
210 215 220
Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro
225 230 235 240
Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr
245 250 255
Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser
260 265 270
Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Lys Pro Arg
275 280 285
Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile
290 295 300
Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn
305 310 315 320
Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys
325 330 335
Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu
340 345 350
Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn Phe
355 360 365
Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala
370 375 380
Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr
385 390 395 400
Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly
405 410 415
Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His
420 425 430
Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys
435 440
<210> 10
<211> 220
<212> PRT
<213> Artificial sequence ()
<400> 10
Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Leu Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asp Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
65 70 75 80
Leu Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln
85 90 95
Gly Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu
100 105 110
Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser
115 120 125
Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn
130 135 140
Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu
145 150 155 160
Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr
180 185 190
Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr
195 200 205
Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
210 215 220

Claims (11)

1. An anti-HBeAg antibody or antigen binding fragment thereof, wherein the antibody comprises the following CDRs:
HCDR1 with an amino acid sequence shown as SEQ ID NO.1, HCDR2 with an amino acid sequence shown as SEQ ID NO.2 and HCDR3 with an amino acid sequence shown as SEQ ID NO. 3; the amino acid sequence is LCDR1 shown in SEQ ID NO.4, the amino acid sequence is LCDR2 shown in SEQ ID NO.5, and the amino acid sequence is LCDR3 shown in SEQ ID NO. 6.
2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody further comprises at least one of a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 8.
3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody further comprises a heavy chain constant region and a light chain constant region; the heavy chain constant region is any one or more of IgG1, igG2, igG3, igG4, igA, igD, igE or IgM, and the light chain constant region is a kappa chain or a lambda chain;
the species of the heavy chain constant region and the light chain constant region are from cattle, horses, dairy cattle, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, turkeys or humans.
4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain of the antibody has the amino acid sequence shown in SEQ ID NO. 9, and the light chain of the antibody has the amino acid sequence shown in SEQ ID NO. 10.
5. The antibody or antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment is selected from the group consisting of Fab, fab ', F (ab') 2 scFv, fv, fd, single chain antibody, diabody, or domain antibody.
6. A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 5.
7. A vector comprising the nucleic acid of claim 6.
8. A cell comprising the nucleic acid of claim 6 or the vector of claim 7.
9. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the nucleic acid according to claim 6, the vector according to claim 7 or the cell according to claim 8 for the preparation of a reagent/kit for the detection/diagnosis of HBV infection.
10. A reagent/kit for detecting/diagnosing HBV infection, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the nucleic acid according to claim 6, the vector according to claim 7, or the cell according to claim 8.
11. A method for detecting HBeAg, which comprises contacting the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 with a sample to be detected.
CN202110861238.3A 2021-07-29 2021-07-29 anti-HBeAg antibody or antigen binding fragment thereof and application thereof Active CN115677853B (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5848856A (en) * 1981-09-17 1983-03-22 Green Cross Corp:The Preparation of specific antibody for hbeag and detection reagent of hbeag
CN1757653A (en) * 2005-10-28 2006-04-12 中国科学院上海生命科学研究院 Anti--HBeAg monoclonal antibody and cell strain, preparation method and purposes
CN112979788A (en) * 2019-12-13 2021-06-18 东莞市朋志生物科技有限公司 Binding protein specifically binding to HBeAg, and reagent and kit for diagnosing HBV infection

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5848856A (en) * 1981-09-17 1983-03-22 Green Cross Corp:The Preparation of specific antibody for hbeag and detection reagent of hbeag
CN1757653A (en) * 2005-10-28 2006-04-12 中国科学院上海生命科学研究院 Anti--HBeAg monoclonal antibody and cell strain, preparation method and purposes
CN112979788A (en) * 2019-12-13 2021-06-18 东莞市朋志生物科技有限公司 Binding protein specifically binding to HBeAg, and reagent and kit for diagnosing HBV infection

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LING CHEN YAN等: "Monoclonal antibody against non-dominant epitopes of HBV e Ag was raised by antigen-antibody co-immunization", 《JOURNAL OF BIOTECHNOLOGY》, vol. 129, no. 4, pages 620 - 627, XP022034273, DOI: 10.1016/j.jbiotec.2007.02.027 *
吴利红: "抗HBeAg不同位点的单克隆抗体杂交瘤细胞株的制备及其应用", 《检验医学》, vol. 4, pages 369 - 372 *

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