Targeting influenza A virus H5N1 high-affinity humanized antibody and application thereof

Abstract

The invention discloses a high-affinity humanized antibody targeting influenza A virus H5N1 and application thereof, amino acid sequences of HCDR1-3 in a heavy chain variable region of the antibody are respectively as shown in SEQ ID NO: 1-3, amino acid sequences of LCDR1-3 in a light chain variable region of the antibody are respectively as shown in SEQ ID NO: 4-6, and experiments verify that the HCDR1-3 in a heavy chain variable region of the antibody has a higher affinity than that in the prior art. The antibody can be specifically combined with hemagglutinin protein of the influenza A virus H5N1, has very high affinity, can be effectively used for detecting the influenza A virus H5N1, and has important application value in development of detection reagents or detection products related to the influenza A virus H5N1.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a high-affinity humanized antibody targeting influenza A virus H5N1 and its applications. Background Art

[0002] Influenza A virus H5N1 is a highly pathogenic subtype of the genus Influenzavirus A in the family Orthomyxoviridae, with hemagglutinin of type 5 and neuraminidase of type 1. This virus has a high lethality rate in poultry, mainly spreads through contact with infected poultry or contaminated environments, and can occasionally infect humans and mammals (such as farmed fur animals, seals, etc.) across species. The incubation period in humans is usually 2 - 4 days, and the initial symptoms are flu-like symptoms such as high fever and cough. In severe patients, the condition progresses rapidly, and complications such as acute respiratory distress syndrome and multiple organ failure can be triggered, with a fatality rate as high as about 50%. The hemagglutinin (HA) protein of influenza A virus H5N1 is a key antigen on the virus surface and has a high subtype specificity.

[0003] Antibodies developed against the HA protein of influenza A virus H5N1 can accurately recognize the unique antigenic epitopes of H5N1 virus, avoid cross-reactions with other influenza virus subtypes (such as H1N1, H3N2) or avian viruses (such as low-pathogenic avian influenza viruses), thereby improving the accuracy of detection results and providing a reliable basis for epidemic prevention and control. Currently, although detection methods for H5N1 (such as RT-PCR) are highly sensitive, they rely on laboratory conditions and cannot meet the requirements for rapid on-site detection. Point-of-care testing (POCT) technology based on H5N1 HA antibodies can make up for this technical defect. Therefore, the development of efficient H5N1 detection tools is a key link in preventing the spread of the virus and reducing the transmission risk. Summary of the Invention

[0004] In order to overcome the technical problems existing in the current field, the present invention has obtained a brand-new high-affinity humanized antibody 323 - 37 targeting influenza A virus H5N1 through a large number of screenings and validations. It can be used for the rapid and accurate detection of influenza A virus H5N1, realizing early detection, early disposal, and early control, and minimizing the threat of influenza A virus H5N1 to human health, the animal industry, and the global economy, which has important significance and wide application value.

[0005] In order to achieve the above invention purposes, the present invention adopts the following technical solutions:

[0006] In the first aspect, the present invention provides a high-affinity antibody targeting the HA protein of influenza A virus H5N1.

[0007] Further, the amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the antibody are shown as SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively;

[0008] The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the antibody are shown as SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0009] Further, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:13 or a sequence having at least 90% homology with SEQ ID NO:13.

[0010] Further, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:14 or a sequence having at least 90% homology with SEQ ID NO:14.

[0011] In the present invention, the influenza A virus H5N1 is a highly pathogenic virus subtype of the genus Orthomyxovirus, family Orthomyxoviridae, and its hemagglutinin (HA) is subtype 5 and its neuraminidase (NA) is subtype 1.

[0012] In some embodiments, antibodies corresponding to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having at least 70% homology with the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are also included within the scope of protection of the present invention, where at least 70% homology includes at least 70% homology, at least 75% homology, at least 80% homology, at least 85% homology, at least 86% homology, at least 87% homology, at least 88% homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology.

[0013] In the present invention, the homology refers to a certain degree of complementarity, which can be partial homology, substantial homology or complete homology. Substantial homology refers to a partially complementary sequence that at least partially inhibits the hybridization of the same sequence with the target nucleic acid. Hybridization experiments (such as Southern or Northern blotting, solution hybridization, etc.) can be used under low stringency conditions to test the inhibition of the hybridization of the completely complementary sequence with the target sequence. Substantially homologous sequences or hybridization probes will compete with and inhibit the binding of completely homologous sequences to the target sequence under low stringency conditions. This does not mean that non-specific binding is allowed under low stringency conditions; low stringency conditions require the interaction between the two sequences to be a specific (selective) interaction.

[0014] In some embodiments, the amino acid sequences corresponding to the HCDR1, HCDR2, and HCDR3 of the present invention are not limited to the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and the amino acid sequences corresponding to the LCDR1, LCDR2, and LCDR3 of the present invention are also not limited to the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. The amino acid sequences or nucleotide sequences of the antibodies corresponding to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 defined by any CDR numbering scheme (existing CDR numbering schemes or newly generated CDR numbering schemes in the future) for the CDR1, CDR2, and CDR3 in the heavy chain variable region shown in SEQ ID NO:13 and the CDR1, CDR2, and CDR3 in the light chain variable region shown in SEQ ID NO:14 are within the scope of protection of the present invention.

[0015] In specific embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to any one of the IMGT numbering scheme, Chothia numbering scheme, Kabat numbering scheme, Martin (enhanced Chothia) numbering scheme, AbM numbering scheme, Aho numbering scheme, and Contact numbering scheme, or any combination of any multiple (two or more) of them. The sequences of the antibodies corresponding to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 defined by the above definition methods are also included within the scope of protection of the present invention.

[0016] In a second aspect, the present invention provides a nucleic acid molecule encoding the antibody as described above in the present invention.

[0017] In some embodiments, the nucleic acid molecule sequence encoding the heavy chain variable region of the antibody of the present invention as described above is shown in SEQ ID NO: 15, and the nucleic acid molecule sequence encoding the light chain variable region of the antibody of the present invention as described above is shown in SEQ ID NO: 16.

[0018] In the present invention, the nucleic acid molecule generally refers to any nucleic acid sequence, for example, any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA, or modified RNA or DNA. It includes but is not limited to: single-stranded and double-stranded DNA, including DNA with single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA with single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA. It also includes triple-stranded regions containing RNA or DNA or both RNA and DNA. Specifically, it includes mRNA, cDNA, and genomic DNA and any fragments thereof. The polynucleotide includes DNA and RNA containing one or more modified bases such as tritiated bases or unusual bases such as inosine. The nucleic acid molecule of the present invention can cover coding or non-coding sequences. It should be understood that every mention of nucleic acid molecule or similar terms herein will include the full-length sequence, and any complementary sequence, fragment, variant, derivative, or modification thereof.

[0019] In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule of the present invention as described above.

[0020] In some embodiments, the vector includes plasmids, virus-derived vectors, phagemids, cosmids, artificial chromosomes.

[0021] In some embodiments, the virus-derived vectors include lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, poxviral vectors, herpesviral vectors, baculoviral vectors.

[0022] In the present invention, there is no particular limitation on the vector expressing the coding sequence of the antibody of the present invention as described above, including but not limited to: bacteria transformed with microbial bodies such as recombinant phages, plasmids, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems transformed with viral expression vectors (such as baculoviruses); plant cells transformed with viral expression vectors (such as cauliflower mosaic virus CaMV, tobacco mosaic virus TMV) or bacterial expression vectors (such as Ti, pBR322 plasmid); or animal cell systems. For bacteria, useful plasmids include pET, pRSET, pTrcHis2, and pBAD plasmids from Invitrogen; pET and pCDF plasmids from Novagen, and DirectorTM plasmids from Sigma-Aldrich. For methanogens, useful plasmids include but are not limited to pME2001, pMV15, and pMP1.

[0023] In a fourth aspect, the present invention provides a host cell comprising the expression vector as described above in the present invention.

[0024] In the present invention, there is no particular limitation on the type of the host cell, and any suitable host cell can be used for the expression of the DNA sequence encoding the antibody as described above in the present invention or the nucleic acid molecule as described above in the present invention, including but not limited to: mammalian cells, plant cells, insect cells, fungal cells or cells derived from bacteria. In some embodiments, the host cell is preferably a mammalian cell.

[0025] In a fifth aspect, the present invention provides an antibody derivative.

[0026] Furthermore, the antibody derivative comprises the antibody as described above.

[0027] In some embodiments, the antibody derivative includes an antibody derivative obtained by conjugating or coupling the antibody as described above in the present invention with a diagnostic agent.

[0028] In some embodiments, the diagnostic agent includes a bioluminescent agent, a chemiluminescent agent, a paramagnetic ion, a radionuclide, an enzyme, a photosensitive diagnostic agent.

[0029] In the present invention, the diagnostic agent is not limited to a bioluminescent agent, a chemiluminescent agent, a paramagnetic ion, a radionuclide, an enzyme, a photosensitive diagnostic agent, and any reagent capable of conjugating or coupling with the antibody as described above in the present invention for the detection of influenza A virus H5N1 is within the protection scope of the present invention.

[0030] In a sixth aspect, the present invention provides a detection reagent or a detection product.

[0031] Furthermore, the detection reagent or the detection product comprises the antibody as described above in the present invention.

[0032] In some embodiments, the detection product includes a detection kit, a test strip, a detection chip.

[0033] In some embodiments, the detection kit further includes a solid-phase carrier, and the antibody is immobilized on the solid-phase carrier (such as a microplate, a coverslip, a microbead) or exists freely. The kit further includes: a detectable moiety capable of connecting with the antibody, and the detectable moiety can be separably present in the kit; and / or a substrate corresponding to the detectable moiety; and / or enzyme-linked immunosorbent reaction reagents, and the reaction reagents include but are not limited to: coating (buffer) solution, washing (buffer) solution, blocking solution, fixing solution, termination solution, chromogenic solution; and / or an instruction manual for describing the method for detecting influenza A virus H5N1 HA protein.

[0034] In addition, the present invention also provides a pharmaceutical composition or a pharmaceutical preparation.

[0035] Furthermore, the pharmaceutical composition or pharmaceutical preparation comprises the antibody as described above in the present invention.

[0036] In some embodiments, the pharmaceutical composition or pharmaceutical preparation provided by the present invention can be administered to a subject in need thereof by any route known in the art, including but not limited to: oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal routes.

[0037] In some embodiments, the dosage forms of the pharmaceutical preparation provided by the present invention include but are not limited to: injections, tablets, pills, dragees, capsules, gels, syrups or suspensions.

[0038] In some embodiments, the pharmaceutical composition or pharmaceutical preparation provided by the present invention may further comprise pharmaceutically acceptable excipients, which include but are not limited to: lactose, sucrose, mannitol, sorbitol, starch, methylcellulose, hydroxypropylmethyl - cellulose, sodium carboxymethylcellulose, gum arabic, tragacanth, gelatin, collagen, cross - linked polyvinylpyrrolidone, agar, sodium alginate, talc, magnesium stearate, fatty oils, polyethylene glycol, carbomer gel, titanium dioxide, edible dyes, dextran, etc.

[0039] In a seventh aspect, the present invention provides any one of the following methods:

[0040] (1) A method for preparing the antibody as described above in the present invention, the method comprising the following steps: culturing the host cell as described above in the present invention and recovering the antibody as described above in the present invention;

[0041] (2) A method for preparing the host cell as described above in the present invention, the method comprising the following steps: introducing the expression vector as described above in the present invention into a host cell to obtain the host cell as described above in the present invention;

[0042] (3) A method for non - diagnostically detecting the influenza A virus H5N1 HA protein, the method comprising the following steps: contacting a sample to be tested with the antibody as described above in the present invention, the antibody derivative as described above in the present invention, the detection reagent or detection product as described above in the present invention, detecting the immune reaction of the sample with the antibody, and determining the expression level of the influenza A virus H5N1 HA protein in the sample.

[0043] In some embodiments, the antibody can be recovered and purified from recombinant cell cultures by methods well known in the art, including but not limited to: ammonium sulfate or ethanol precipitation, acid extraction, Protein A affinity chromatography, Protein G affinity chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography (HPLC) can be used for purification.

[0044] The antibodies of the present invention include products of natural purification, products of chemical synthesis methods, and products produced by recombinant techniques from prokaryotic and eukaryotic hosts, and the eukaryotic hosts include but are not limited to: yeast, higher plants, insects, and mammals. The antibodies of the present invention can be glycosylated or can be non-glycosylated. Such methods are described in many standard laboratory manuals.

[0045] In an eighth aspect, the present invention provides the following applications in any aspect:

[0046] (1) Use of the antibody as described above in the present invention, the nucleic acid molecule as described above in the present invention, the expression vector as described above in the present invention, and / or the host cell as described above in the present invention in the preparation of antibody derivatives for detecting influenza A virus H5N1 HA protein;

[0047] (2) Use of the antibody as described above in the present invention, the nucleic acid molecule as described above in the present invention, the expression vector as described above in the present invention, the host cell as described above in the present invention, and / or the antibody derivative as described above in the present invention in the preparation of detection reagents for detecting influenza A virus H5N1 HA protein;

[0048] (3) Use of the antibody as described above in the present invention, the nucleic acid molecule as described above in the present invention, the expression vector as described above in the present invention, the host cell as described above in the present invention, the antibody derivative as described above in the present invention, and / or the detection reagent as described above in the present invention in the preparation of detection products for detecting influenza A virus H5N1 HA protein;

[0049] (4) Use of the antibody as described above in the present invention, the nucleic acid molecule as described above in the present invention, the expression vector as described above in the present invention, the host cell as described above in the present invention, the antibody derivative as described above in the present invention, and / or the detection reagent or detection product as described above in the present invention in the detection of influenza A virus H5N1 HA protein for non-diagnostic purposes;

[0050] (5) Use of the antibody as described above in the present invention, the nucleic acid molecule as described above in the present invention, the expression vector as described above in the present invention, the host cell as described above in the present invention, the antibody derivative as described above in the present invention, and / or the detection reagent or detection product as described above in the present invention in the preparation of products for diagnosing influenza A virus H5N1 infection diseases.

[0051] In addition, the present invention also provides a method for diagnosing H5N1 avian influenza virus infection disease.

[0052] Furthermore, the method comprises the following steps: detecting a sample to be tested from a subject by using the antibody as described above in the present invention, the antibody derivative as described above in the present invention, and / or the detection reagent or detection product as described above in the present invention, and detecting the presence of H5N1 HA protein of avian influenza virus in the sample to be tested from the subject through an antigen-antibody reaction, so as to diagnose whether the subject has H5N1 avian influenza virus infection disease.

[0053] In some embodiments, the present invention places no particular limitation on the sample to be tested, and the sample to be tested is derived from a clinical sample of a subject in need, including but not limited to: cells, tissues, body fluids, such as: skin; mucosa; blood; blood derivatives, such as serum; extracted bile; tissue taken by biopsy or surgery, including for example unfixed, frozen, fixed in formalin and / or embedded in paraffin; tears; milk; dandruff; surface washings; urine; sputum; cerebrospinal fluid; prostatic fluid; pus; bone marrow aspirate; middle ear effusion; bronchoalveolar lavage fluid; sputum or saliva. In other embodiments, the sample to be tested may also be an environmental sample or a food sample.

[0054] In some embodiments, the subjects include but are not limited to: humans, avians, non-human mammals. Exemplarily, the avians include but are not limited to: chickens, ducks, geese, quails, wild geese, pigeons, peacocks, francolins, and the non-human mammals include but are not limited to: pigs, cats, dogs, minks, foxes, seals, sea lions, rats.

[0055] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0056] The present invention provides a novel high-affinity humanized antibody targeting H5N1 avian influenza virus for the field. It is experimentally verified that the antibody can specifically bind to the hemagglutinin protein of H5N1 avian influenza virus, has a very high affinity, can be effectively used for detecting H5N1 avian influenza virus, and has important application value in the development of detection reagents or detection products related to H5N1 avian influenza virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 It is a SDS-PAGE electrophoresis result diagram, wherein, from left to right are protein molecular weight Marker, reduced 323-37 antibody, and non-reduced 323-37 antibody in sequence;

[0058] Figure 2 It is a result diagram corresponding to the 323-37 antibody having a relatively high affinity for H5N1 HA protein;

[0059] Figure 3 It is a result graph corresponding to the affinity constant of the 323-37 antibody and H5N1 HA detected by SPR;

[0060] Figure 4 It is a result graph corresponding to the high specificity of the 323-37 antibody for the H5N1 HA protein. Detailed implementation manners

[0061] The present invention will be further described below in conjunction with specific embodiments. The following specific embodiments are only used to explain the present invention and should not be construed as limiting the present invention. Those of ordinary skill in the art can understand that: without departing from the principles and purposes of the present invention, various changes, modifications, substitutions and variations can be made to these embodiments. The scope of the present invention is defined by the claims and their equivalents. The experimental consumables, reagents and raw materials used in the present invention are easily obtained by those of ordinary skill in the art. Unless otherwise specified, they can all be obtained from commercial channels. The experimental methods without specific conditions noted in the present invention are usually carried out under conventional conditions or according to the conditions recommended by the manufacturers. In particular, the following embodiments are only used to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and their results described in the following embodiments are only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims.

[0062] Example 1 Screening of antibodies targeting the Hemagglutinin (HA) protein of avian influenza virus H5N1

[0063] 1. Experimental method

[0064] After immunizing mice with the HA antigen of H5N1 (purchased from Sino Biological Inc.) multiple times, single cell suspensions were prepared from the spleens of the mice, and antigen-specific B cells were obtained by flow sorting.

[0065] The obtained antigen-specific B cells were sent to Yiyan Technology to construct a single B cell library using the 10X technology platform. During the library construction process, the immunoglobulin genes (genes encoding antibodies) in each B cell were sequenced to obtain a large amount of antibody gene sequence information.

[0066] 2. Experimental results

[0067] The 323-37 antibody targeting the Hemagglutinin (HA) protein of avian influenza virus H5N1 was screened. The sequence information of the 323-37 antibody is as follows:

[0068] Amino acid sequence of CDR1 in the heavy chain variable region: GFTFRIH (SEQ ID NO: 1);

[0069] Heavy chain variable region CDR2 amino acid sequence: SGSGGN (SEQ ID NO:2);

[0070] Heavy chain variable region CDR3 amino acid sequence: CAKLGWFDGFDIW (SEQ ID NO:3);

[0071] Light chain variable region CDR1 amino acid sequence: RSSTGAVTTSNYAN (SEQ ID NO:4);

[0072] Light chain variable region CDR2 amino acid sequence: GTNNRAP (SEQ ID NO:5);

[0073] Light chain variable region CDR3 amino acid sequence: CALWYSNHWVF (SEQ ID NO:6);

[0074] Heavy chain variable region CDR1 nucleotide sequence: GGATTCACTTTCAGAATTCAT (SEQ ID NO:7);

[0075] Heavy chain variable region CDR2 nucleotide sequence: AGTGGTAGTGGTGGTAAC (SEQ ID NO:8);

[0076] Heavy chain variable region CDR3 nucleotide sequence: TGTGCGAAACTGGGGTGGTTTGATGGTTTTGATATTTGG (SEQ ID NO:9);

[0077] Light chain variable region CDR1 nucleotide sequence: CGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGCCAAC (SEQ ID NO:10);

[0078] Light chain variable region CDR2 nucleotide sequence: GGAACCAACAACCGAGCTCCA (SEQ ID NO:11);

[0079] Light chain variable region CDR3 nucleotide sequence: TGTGCTCTATGGTACAGCAACCATTGGGTGTTC (SEQ ID NO:12);

[0080] Heavy chain variable region amino acid sequence: EVQLVESGGGLVQLGGSLRVSCAASGFTFRIHAMTWVRQAPGRGLEWVSTISGSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVHYCAKLGWFDGFDIWGQGTMVTVSS (SEQ ID NO:13);

[0081] Amino acid sequence of the light chain variable region: QAVVTQESALITSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAVLTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVL (SEQ ID NO:14);

[0082] Nucleotide sequence of the heavy chain variable region: GAGGTCCAGTTGGTGGAGTCTGGGGGAGGCTTGGTACAGCTTGGGGGGTCCCTGAGAGTCTCCTGTGCAGCCTCTGGATTCACTTTCAGAATTCATGCCATGACTTGGGTCCGCCAGGCTCCAGGGAGGGGGCTGGAGTGGGTCTCAACTATTAGTGGTAGTGGTGGTAACACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTACATTACTGTGCGAAACTGGGGTGGTTTGATGGTTTTGATATTTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAG (SEQ ID NO:15);

[0083] Nucleotide sequence of the light chain variable region: CAGGCTGTTGTGACTCAGGAATCTGCACTCATCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGGAACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGTCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAG (SEQ ID NO:16).

[0084] Example 2 Preparation of the 323-37 Antibody Targeting the HA Protein of Avian Influenza Virus H5N1

[0085] 1. Experimental methods

[0086] (1) The DNA sequences of VH and VL of the 323-37 antibody obtained by sequencing in Example 1 were sent to Genewiz for synthesis, and the synthesized sequences were ligated onto a laboratory expression vector by enzymatic digestion and ligation.

[0087] (2) The expression vector was transfected into CHO mammalian cells for expression. The expression supernatant was collected.

[0088] (3) The antibody in the CHO cell culture supernatant was purified using Protein A on an AKTA protein purifier.

[0089] (4) Elution was carried out and collected using 3.5 mM citrate buffer, and the pH was immediately adjusted to approximately 7-8 with 1 M Tris at pH 11.5 after collection was completed.

[0090] (5) After ultrafiltration and concentration using a pH 7.2, 0.01 mol / L PBS in an ultrafiltration tube, it was filtered through a 0.22 μm filter membrane to sterilize.

[0091] (6) SDS-PAGE experiments were used to detect the expression and purification of the antibody, and the BCA method was used to detect the concentration of the purified antibody, which was stored at 4°C.

[0092] 2. Experimental results

[0093] The experimental results are as Figure 1 shown. The results show that a monoclonal antibody (323-37 antibody) targeting the avian influenza virus H5N1 HA protein was successfully prepared in this example.

[0094] Example 3 ELISA detection of the binding of the 323-37 antibody targeting the avian influenza virus H5N1 HA protein to H5N1 HA

[0095] 1. Experimental methods

[0096] (1) The antigen was diluted to 0.25 mg / mL with sterile injection water in the cell culture hood.

[0097] (2) The HA antigen was diluted to 1 μg / mL with the coating buffer, and 100 μL / well of the antigen was coated, at 37°C for 2 h.

[0098] (3) The plate was washed three times with a plate washer and patted dry on a paper towel.

[0099] (4) 4% skim milk powder was prepared with PBS, and 250 μL / well was used for blocking, at 37°C for 1 h.

[0100] (5)The candidate 323-37 antibody was diluted with PBS to an initial concentration of 10 μg / mL.

[0101] (6)Twelve concentrations were serially diluted in a 96-well deep-well plate, starting from 10 μg / mL and diluted three-fold serially. Except for the first column, 250 μL of PBS was added to each well. 125 μL of the 10 μg / mL antibody was aspirated from the first column and added to the wells containing 250 μL of PBS in the second column, and mixed well by pipetting. Dilution was carried out successively to the 12th well, with a three-fold serial dilution.

[0102] (7)The plate washer was taken out of the incubator in step (4), and the plate was washed three times and patted dry on a paper towel.

[0103] (8)The diluted antibodies of different concentrations in each row were added to the blocked plate at one time using a 100 μL multi-channel pipette. Incubate at 37 °C for 1 h.

[0104] (9)The plate was washed three times with a plate washer and patted dry on a paper towel.

[0105] (10)GAH-HRP was diluted at 1:6000 and 100 μL / well was added to the plate. Incubate at 37 °C for 30 min.

[0106] (11)The plate was washed three times with a plate washer and patted dry on a paper towel.

[0107] (12)Prepare the TMB working solution and add 100 μL / well to the plate after incubating with the primary antibody. After obvious color gradient appeared, terminate the color development with 100 μL / well of 0.5 M sulfuric acid solution.

[0108] (13)Measure the OD450 value with an ELISA reader within 5 min, use graphad prism to plot the binding curve, and calculate the EC50 value.

[0109] 2. Experimental Results

[0110] The experimental results are as Figure 2 shown. The results show that the 323-37 antibody targeting the HA protein of avian influenza virus H5N1 has a relatively high affinity (in the nM level) for binding to the HA protein.

[0111] Example 4 SPR Detection of the Affinity Constant between the 323-37 Antibody Targeting the HA Protein of Avian Influenza Virus H5N1 and H5N1 HA

[0112] 1. Experimental Method

[0113] The binding kinetics of the 323-37 antibody was determined using a Biacore T200 instrument (Cytiva) in HBS-EP mobile phase (BR100669, Cytiva) at 25 °C. First, the carboxyl groups on the CM5 sensor chip matrix were activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to generate reactive esters that can react with amino groups. The HA antigen was diluted to 1 μg / mL in 10 mM sodium acetate buffer at pH 5.0 and covalently coupled to a immobilized level of approximately 180 response units (RU). Subsequently, unreacted reactive esters were blocked with ethanolamine.

[0114] Subsequently, the binding of HA to the 323-37 antibody was analyzed using a multi-cycle kinetics method. The concentration range of the 323-37 antibody used was 3.125 - 50 nM, with a two-fold dilution. The binding phase lasted for 180 seconds, the dissociation phase lasted for 300 seconds, the flow rate was 30 μL / min, and finally regeneration treatment was performed. The binding kinetic parameters were calculated by a global kinetic fitting model (1:1 Langmuir) (Biacore T200 Evaluation Software 3.1, Cytiva).

[0115] 2. Experimental Results

[0116] The experimental results are as Figure 3 shown. The results show that the 323-37 antibody targeting the HA protein of avian influenza virus H5N1 has a high affinity constant KD of 10 to the power of -10 (KD (M) = 4.249E-10) for binding to the HA antigen, indicating strong affinity.

[0117] Example 5 Detection of the Specificity of the 323-37 Antibody Targeting the HA Protein of Avian Influenza Virus H5N1 to H5N1 HA

[0118] 1. Experimental Method

[0119] The experimental method was the same as that in Example 3.

[0120] 2. Experimental Results

[0121] The experimental results are as Figure 4 shown. The results show that the 323-37 antibody targeting the HA protein of avian influenza virus H5N1 cannot bind to the HA antigen of avian influenza virus H7N9, indicating that the 323-37 antibody targeting the HA protein of avian influenza virus H5N1 screened and prepared in the present invention can specifically bind to the HA antigen of avian influenza virus H5N1.

[0122] The experimental results of the above embodiments show that the 323-37 antibody targeting the HA protein of avian influenza virus H5N1 screened and prepared by the present invention can specifically bind to the avian influenza virus H5N1 HA antigen, and has good specificity and affinity, and can be used in the development of detection or auxiliary detection products related to the avian influenza virus H5N1 HA protein.

Claims

1. A high-affinity antibody targeting the HA protein of influenza A virus H5N1, characterized in that, The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the antibody are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the antibody are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

2. The antibody according to claim 1, wherein The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:13 or a sequence having at least 90% homology with SEQ ID NO:

13.

3. The antibody according to claim 1, wherein The amino acid sequence of the light chain variable region is shown in SEQ ID NO:14 or a sequence having at least 90% homology with SEQ ID NO:

14.

4. A nucleic acid molecule encoding the antibody according to any one of claims 1-3.

5. An expression vector comprising the nucleic acid molecule according to claim 4.

6. A host cell comprising the expression vector according to claim 5.

7. An antibody derivative, characterized in that, The antibody derivative comprises the antibody according to any one of claims 1-3.

8. A detection reagent or detection product, characterized in that, The detection reagent or detection product comprises the antibody according to any one of claims 1-3.

9. Any one of the following methods: (1) A method for preparing the antibody according to any one of claims 1-3, the method comprising the following steps: culturing the host cell according to claim 6, and recovering the antibody according to any one of claims 1-3; (2) A method for preparing the host cell according to claim 6, the method comprising the following steps: introducing the expression vector according to claim 5 into a host cell to obtain the host cell according to claim 6; (3) A method for non-diagnostically detecting the influenza A virus H5N1 HA protein, the method comprising the following steps: contacting a sample to be tested with the antibody according to any one of claims 1-3, the antibody derivative according to claim 7, the detection reagent or detection product according to claim 8, detecting the immune reaction between the sample and the antibody, and determining the expression level of the influenza A virus H5N1 HA protein in the sample.

10. Any one of the following applications: (1) Application of the antibody according to any one of claims 1-3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, and / or the host cell according to claim 6 in the preparation of an antibody derivative for detecting the influenza A virus H5N1 HA protein; (2) Application of the antibody according to any one of claims 1-3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, the host cell according to claim 6, and / or the antibody derivative according to claim 7 in the preparation of a detection reagent for detecting the influenza A virus H5N1 HA protein; (3) Application of the antibody according to any one of claims 1-3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, the host cell according to claim 6, the antibody derivative according to claim 7, and / or the detection reagent according to claim 8 in the preparation of a detection product for detecting the influenza A virus H5N1 HA protein; (4)Use of the antibody according to any one of claims 1-3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, the host cell according to claim 6, the antibody derivative according to claim 7 and / or the detection reagent or detection product according to claim 8 for detecting influenza A virus H5N1 HA protein for non-diagnostic purposes; (5)Use of the antibody according to any one of claims 1-3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, the host cell according to claim 6, the antibody derivative according to claim 7 and / or the detection reagent or detection product according to claim 8 in the preparation of a product for diagnosing influenza A virus H5N1 infection diseases.

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