Targeting influenza a virus h5n1 high affinity humanized antibodies and uses thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to a high-affinity humanized antibody targeting influenza A virus H5N1 and application thereof. BACKGROUND

[0002] Influenza A virus H5N1 is a highly pathogenic subtype of Orthomyxoviridae influenza A virus, and its hemagglutinin is type 5 and its neuraminidase is type 1. The virus has a high mortality rate for birds, and is mainly transmitted through contact with sick birds or contaminated environment, and occasionally can infect humans and mammals (such as farmed fur animals, seals, etc.) across species. The incubation period of human infection is usually 2-4 days, and the early symptoms are high fever, cough and other flu-like symptoms. Severe patients progress rapidly and can cause acute respiratory distress syndrome, multiple organ failure and other complications, with a mortality rate of about 50%. The hemagglutinin (HA) protein of influenza A virus H5N1 is a key antigen on the surface of the virus, and has high subtype specificity.

[0003] The antibody developed for the HA protein of influenza A virus H5N1 can accurately recognize the unique antigen epitope of H5N1 virus, avoiding cross-reaction with other influenza virus subtypes (such as H1N1, H3N2) or avian viruses (such as low pathogenic avian influenza virus), thereby improving the accuracy of the detection results and providing a reliable basis for epidemic prevention. At present, although the detection method (such as RT-PCR) for H5N1 has high sensitivity, it depends on laboratory conditions and cannot meet the needs of on-site rapid detection. The point-of-care testing (POCT) technology based on H5N1 HA antibody 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 risk of transmission. SUMMARY

[0004] In order to overcome the technical problems existing in the prior art, the present application obtains a novel high-affinity humanized antibody 323-37 targeting influenza A virus H5N1 through a large number of screening and verification, which can be used for rapid and accurate detection of influenza A virus H5N1, realizes early detection, early disposal and early control, and maximally reduces the threat of influenza A virus H5N1 to human health, animal industry and global economy, and has important significance and wide application value.

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

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

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

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

[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 application, the influenza A virus H5N1 is a highly pathogenic virus subtype of Orthomyxoviridae Influenza A virus genus, and the hemagglutinin (HA) thereof is subtype 5 and the neuraminidase (NA) is subtype 1.

[0012] In some embodiments, the antibodies corresponding to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 having at least 70% homology with the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 are also included in the protection scope of the present application, wherein the 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 application, the homology refers to a certain degree of complementarity. It can be partially homologous, substantially homologous or completely homologous. Substantially homologous refers to a partially complementary sequence that at least partially inhibits the hybridization of the identical sequence to the target nucleic acid. The inhibition of the hybridization of the completely complementary sequence to the target sequence can be tested by using a hybridization experiment (Southern or northern blotting, solution hybridization, etc.) under low stringency conditions. The substantially homologous sequence or hybridization probe will compete with and inhibit the binding of the completely homologous sequence to the target sequence under low stringency conditions. This is not to say that the low stringency conditions allow non-specific binding; the low stringency conditions require that the binding of the two sequences to each other is a specific (selective) interaction.

[0014] In some embodiments, the HCDR1, HCDR2, HCDR3 corresponding amino acid sequences of the present application are not limited to the amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and the LCDR1, LCDR2, LCDR3 corresponding amino acid sequences of the present application are not limited to the amino acid sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6. The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 corresponding to the antibody amino acid sequences or nucleotide sequences obtained by defining the CDR1, CDR2, CDR3 in the heavy chain variable region as shown in SEQ ID NO: 13 and defining the CDR1, CDR2, CDR3 in the light chain variable region as shown in SEQ ID NO: 14 using any CDR numbering scheme (existing CDR numbering scheme or new CDR numbering scheme to be developed in the future) are all within the scope of the present application.

[0015] In specific embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, 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, Contact numbering scheme, or any combination (two or more) thereof, and the sequences of the antibodies corresponding to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 defined by the above definition methods are also included in the scope of the present application.

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

[0017] In some embodiments, the nucleic acid molecule encoding the heavy chain variable region of the antibody of the application as previously described has the sequence as set forth in SEQ ID NO: 15, and the nucleic acid molecule encoding the light chain variable region of the antibody of the application as previously described has the sequence as set forth in SEQ ID NO: 16.

[0018] In the present application, the nucleic acid molecule generally refers to any nucleic acid sequence, for example, any polynucleotide or polydeoxynucleotide, which can be unmodified RNA or DNA, or modified RNA or DNA. This includes, but is not limited to, single- and double-stranded DNA, including supercoiled, circular, linear, partial or completed double-stranded DNA, single- and double-stranded RNA, and RNA including single- and double-stranded regions. Hybrid molecules comprising DNA and RNA are also included. Triple-stranded regions like those which occur in triple helix formation are also included. In particular, mRNA, cDNA, and genomic DNA are included, as are fragments of any of the foregoing. The polynucleotide includes DNA and RNA comprising one or more modified bases, such as tritiated bases or unusual bases, such as inosine. The nucleic acid molecule of the present application can encompass coding or non-coding sequences. It should be understood that each reference to a nucleic acid molecule or similar term herein will include the full-length sequence, as well as any complementary sequence, fragment, variation, derivative, or variant thereof.

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

[0020] In some embodiments, the vector includes a plasmid, a viral-derived vector, a phagemid, a cosmid, an artificial chromosome.

[0021] In some embodiments, the viral-derived vector includes a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a poxvirus vector, a herpesvirus vector, a baculovirus vector.

[0022] In the present application, the vector expressing the coding sequence of the antibody of the application as previously described is not particularly limited, and includes, but is not limited to, a microbial body such as a bacterium transformed with a recombinant bacteriophage, a plasmid, or a cosmid DNA expression vector; a yeast transformed with a yeast expression vector; an insect cell system transformed with a viral expression vector (e.g., baculovirus); a plant cell transformed with a viral expression vector (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or a bacterial expression vector (e.g., Ti, pBR322 plasmid); or an animal cell system. For bacteria, useful plasmids include pET, pRSET, pTrcHis2, and pBAD plasmids from Invitrogen; pET and pCDF plasmids from Novagen; and Director™ 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 application provides a host cell comprising the expression vector of the present application as described above.

[0024] In the present application, the type of the host cell is not particularly limited, and any suitable host cell can be used for the expression of the DNA sequence encoding the antibody of the present application as described above or the nucleic acid molecule of the present application as described above, including but not limited to: a mammalian cell, a plant cell, an insect cell, a fungal cell or a cell of bacterial origin. In some embodiments, the host cell is preferably a mammalian cell.

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

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

[0027] In some embodiments, the antibody derivative includes an antibody derivative obtained by coupling or conjugating the antibody of the present application as described above 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 application, 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 agent capable of being coupled or conjugated with the antibody of the present application as described above for the detection of the influenza A virus H5N1 is within the protection scope of the present application.

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

[0031] Further, the detection reagent or the detection product comprises the antibody of the present application as described above.

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

[0033] In some embodiments, the detection kit further comprises a solid phase carrier, and the antibody is immobilized on the solid phase carrier (such as a multi-well plate, a cover glass, a microbead) or is free. The kit further comprises: a detectable moiety capable of being linked to the antibody, the detectable moiety being separably present in the kit; and / or a substrate corresponding to the detectable moiety; and / or an enzyme-linked immunoassay reagent, including but not limited to: a coating (buffer) solution, a washing (buffer) solution, a blocking solution, a fixing solution, a termination solution, a color developing solution; and / or an instruction manual for the method of detecting the influenza A virus H5N1 HA protein.

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

[0035] Further, the pharmaceutical composition or the pharmaceutical preparation comprises the antibody of the present application as previously described.

[0036] In some embodiments, the pharmaceutical composition or the pharmaceutical preparation of the present application can be administered to a subject in need thereof by any route known in the art, including but not limited to orally, intravenously, intramuscularly, intra-arterially, intramedullary, intrathecally, intraventricularly, transdermally, subcutaneously, intraperitoneally, intranasally, enterally, topically, sublingually or rectally.

[0037] In some embodiments, the dosage form of the pharmaceutical preparation of the present application includes but is not limited to injection, tablet, pill, sugar-coated tablet, capsule, gel, syrup or suspension.

[0038] In some embodiments, the pharmaceutical composition or the pharmaceutical preparation of the present application can further comprise a pharmaceutically acceptable excipient, which includes but is not limited to lactose, sucrose, mannitol, sorbitol, starch, methyl cellulose, hydroxypropyl methyl-cellulose, sodium carboxymethyl cellulose, gum arabic, tragacanth gum, gelatin, collagen, cross-linked polyvinyl pyrrolidone, agar, sodium alginate, talc, magnesium stearate, fatty oil, polyethylene glycol, carbomer gel, titanium dioxide, edible dye, dextran, etc.

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

[0040] (1) a method for preparing the antibody of the present application as previously described, which comprises the following steps: culturing the host cell of the present application as previously described, and recovering the antibody of the present application as previously described;

[0041] (2) a method for preparing the host cell of the present application as previously described, which comprises the following steps: introducing the expression vector of the present application as previously described into a host cell to obtain the host cell of the present application as previously described;

[0042] (3) a method for detecting the HA protein of the influenza A virus H5N1 for non-diagnostic purposes, which comprises the following steps: contacting a sample to be tested with the antibody of the present application as previously described, the derivative of the antibody of the present application as previously described, the detection reagent or the detection product of the present application as previously described, detecting the immunoreaction between the sample and the antibody, and determining the expression level of the HA protein of the influenza A virus H5N1 in the sample.

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

[0044] The antibodies of the present application include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from prokaryotic or eukaryotic host cells, including, but not limited to, yeast, higher plants, insects, and mammals. The antibodies of the present application can be glycosylated or can be non-glycosylated. Such methods are described in many standard laboratory manuals.

[0045] In an eighth aspect, the present application provides use of any of the following:

[0046] (1) use of the antibody of the present application as previously described, the nucleic acid molecule of the present application as previously described, the expression vector of the present application as previously described, and / or the host cell of the present application as previously described in the preparation of an antibody derivative for detecting the HA protein of the influenza A virus H5N1;

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

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

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

[0050] (5) use of the antibody of the present application as previously described, the nucleic acid molecule of the present application as previously described, the expression vector of the present application as previously described, the host cell of the present application as previously described, the antibody derivative of the present application as previously described, and / or the detection reagent or detection product of the present application as previously described in the preparation of a product for diagnosing the disease of the influenza A virus H5N1 infection.

[0051] Further, the present application also provides a method for diagnosing the influenza A virus H5N1 infection disease.

[0052] Further, the method comprises the following steps: using the antibody as described above, the antibody derivative as described above and / or the detection reagent or detection product as described above to detect the sample to be detected from the subject, and detecting the presence of the influenza A virus H5N1 HA protein in the sample to be detected from the subject by antigen-antibody reaction, so as to diagnose whether the subject has the influenza A virus H5N1 infection disease.

[0053] In some embodiments, the sample to be detected is not particularly limited, and the sample to be detected is a clinical sample from 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; tissues removed by biopsy or surgery, including, for example, un-fixed, 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; sputum or saliva. In other embodiments, the sample to be detected can also be an environmental sample or a food sample.

[0054] In some embodiments, the subject includes but is not limited to: human, avian, non-human mammal. Exemplarily, the avian includes but is not limited to: chicken, duck, goose, quail, goose, pigeon, peacock, partridge, and the non-human mammal includes but is not limited to: pig, cat, dog, mink, fox, seal, sea lion, mouse.

[0055] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0056] The present application provides a novel high-affinity humanized antibody targeting the influenza A virus H5N1 for the field, and it is found by experiments that the antibody can specifically bind to the 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 the development of detection reagents or detection products related to the influenza A virus H5N1. BRIEF DESCRIPTION OF DRAWINGS

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

[0058] Figure 2 It is a result chart corresponding to the high affinity of 323-37 antibody to H5N1 HA protein;

[0059] Figure 3 The result graph corresponding to the affinity constant of the 323-37 antibody to the H5N1 HA protein detected by SPR;

[0060] Figure 4 The result graph corresponding to the high specificity of the 323-37 antibody to the H5N1 HA protein. DETAILED DESCRIPTION

[0061] The application will be further described below in conjunction with specific examples. The following specific examples are only used to explain the application and cannot be understood as a limitation to the application. Those skilled in the art can understand that various changes, modifications, replacements and variations can be made to the examples without departing from the principles and the purpose of the application, and the scope of the application is defined by the claims and their equivalents. The experimental consumables, reagents and raw materials used in the application are easily obtained by those skilled in the art, and can be obtained from commercial channels if not otherwise specified, and the experimental methods not specified in the application are usually implemented according to the conventional conditions or the conditions suggested by the manufacturers. In particular, the following examples are only used to illustrate the application and should not limit the scope of the application in any way. It should be noted that the experimental conditions and the results described in the following examples are only used to illustrate the application and should not and will not limit the application described in detail in the claims.

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

[0063] 1. Experimental method

[0064] After the mice were immunized multiple times with the HA antigen of H5N1 (purchased from Yiqiao God State), the single-cell suspension was prepared from the spleen of the mice, and the antigen-specific B cells were obtained by flow sorting.

[0065] The obtained antigen-specific B cells were sent to Yijian Technology Co., Ltd. to sequence and construct a single B cell library using the 10X technology platform. During the construction of the library, 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 H5N1 HA protein of avian influenza virus was screened, and the sequence information of the 323-37 antibody is as follows:

[0068] Amino acid sequence of heavy chain variable region CDR1: 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] Heavy chain variable region amino acid sequence: QAVVTQESALITSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAVLTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVL (SEQ ID NO: 14);

[0082] Heavy chain variable region nucleotide sequence: GAGGTCCAGTTGGTGGAGTCTGGGGGAGGCTTGGTACAGCTTGGGGGGTCCCTGAGAGTCTCCTGTGCAGCCTCTGGATTCACTTTCAGAATTCATGCCATGACTTGGGTCCGCCAGGCTCCAGGGAGGGGGCTGGAGTGGGTCTCAACTATTAGTGGTAGTGGTGGTAACACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTACATTACTGTGCGAAACTGGGGTGGTTTGATGGTTTTGATATTTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAG (SEQ ID NO: 15);

[0083] Light chain variable region nucleotide sequence: CAGGCTGTTGTGACTCAGGAATCTGCACTCATCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGGAACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGTCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAG (SEQ ID NO: 16).

[0084] Example 2. Production of 323-37 antibody targeting avian influenza virus H5N1 HA protein

[0085] 1. Experimental method

[0086] (1) The DNA sequences of the VH and VL of the 323-37 antibody obtained by sequencing in Example 1 were sent to Jinweizhi for synthesis, and the synthesized sequences were loaded on the laboratory expression vector by means of enzyme 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 on an AKTA protein purification instrument using Protein A.

[0089] (4) Elution collection was performed with 3.5 mM citrate buffer, and immediately after completion of the collection, the pH was adjusted to about 7-8 with 1 M Tris at pH 11.5.

[0090] (5) After ultrafiltration concentration in an ultrafiltration tube with PBS at pH 7.2, 0.01 mol / L, 0.22 μm filter membrane was used for sterilization.

[0091] (6) The expression and purification of the antibody were detected by SDS-PAGE experiment, and the purified antibody concentration was detected by BCA method, and stored at 4°C.

[0092] 2. Experimental results

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

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

[0095] 1. Experimental method

[0096] (1) The antigen was diluted to 0.25 mg / mL with sterile water for injection in a cell stand.

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

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

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

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

[0101] (6) Gradient dilution of 12 concentrations in a 96 deep well plate, starting from 10 μg / mL, three-fold gradient dilution, 250 μL PBS was added to each well except the first column, 125 μL of 10 μg / mL antibody was pipetted from the first column into the second column containing 250 μL PBS, mixed well by blowing, and sequentially diluted to the 12th column, three-fold gradient dilution.

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

[0103] (8) 100 μL of each row of different concentrations of diluted antibody was added to the blocked plate at one time. 37°C, 1 h.

[0104] (9) The plate washer was washed three times, and dried on paper towel.

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

[0106] (11) The plate washer was washed three times, and dried on paper towel.

[0107] (12) TMB working solution was prepared, 100 μL / well was added to the plate after incubation of the primary antibody, and 0.5 M sulfuric acid solution 100 μL / well was used to terminate the color development after obvious color gradient was shown.

[0108] (13) The OD450 value was measured within 5 min using an enzyme label instrument, the binding curve was drawn using graphad prism, and the EC50 value was calculated.

[0109] 2. Experimental results

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

[0111] Example 4 SPR detection of the affinity constant of 323-37 antibody targeting H5N1 HA protein of avian influenza virus to H5N1 HA

[0112] 1. Experimental method

[0113] The binding kinetics of the 323-37 antibody were determined using a Biacore T200 instrument (Cytiva) at 25 °C in HBS-EP mobile phase (BR100669, Cytiva). 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 an active ester that reacts 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 an immobilization level of approximately 180 response units (RU). Subsequently, the unreacted active ester was blocked with ethanolamine.

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

[0115] 2. Experimental Results

[0116] Experimental results are as follows Figure 3 As shown, the results indicate that the 323-37 antibody targeting the HA protein of avian influenza virus H5N1 has an affinity constant KD of up to 10 to the power of -10 (KD (M) = 4.249E-10) for binding with the HA antigen, indicating strong affinity.

[0117] Example 5: Detection of the specificity of 323-37 antibody targeting the H5N1 HA protein of avian influenza virus with H5N1 HA.

[0118] 1. Experimental Methods

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

[0120] 2. Experimental Results

[0121] Experimental results are as follows Figure 4 As shown, the results indicate that the 323-37 antibody targeting the H5N1 HA protein of avian influenza virus cannot bind to the H7N9 HA antigen of avian influenza virus, which means that the 323-37 antibody targeting the H5N1 HA protein of avian influenza virus screened and prepared in this invention can specifically bind to the H5N1 HA antigen of avian influenza virus.

[0122] The experimental results of the above examples show that the 323-37 antibody targeting the avian influenza virus H5N1 HA protein screened and prepared by the application 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 influenza A virus H5N1 HA protein, characterized in that, the amino acid sequences of HCDR1, HCDR2, HCDR3 in the heavy chain variable region of the antibody are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 respectively; the amino acid sequences of LCDR1, LCDR2, LCDR3 in the light chain variable region of the antibody are shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 respectively.

2. The antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

13.

3. The antibody of claim 1, wherein the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

14.

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

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

6. A host cell comprising the expression vector of claim 5.

7. A test reagent, characterized by the detection reagent comprises the antibody of any one of claims 1-3.

8. A test product, characterized in that the detection product comprises the antibody of any one of claims 1-3.

9. A method for preparing the antibody of any one of claims 1-3, the method comprising the steps of culturing the host cell of claim 6, and recovering the antibody of any one of claims 1-3.

10. A method for preparing the host cell of claim 6, the method comprising the steps of introducing the expression vector of claim 5 into a host cell to obtain the host cell of claim 6.

11. A method for detecting the influenza A virus H5N1 HA protein for non-diagnostic purposes, the method comprising the steps of contacting a sample to be tested with the antibody of any one of claims 1-3, the detection reagent of claim 7, or the detection product of claim 8, detecting the immunoreaction of the sample with the antibody, and determining the expression level of the influenza A virus H5N1 HA protein in the sample.

12. Use of the antibody of any one of claims 1-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, and / or the host cell of claim 6 in the preparation of a detection reagent for detecting the influenza A virus H5N1 HA protein.

13. Use of the antibody of any one of claims 1-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the host cell of claim 6, and / or the detection reagent of claim 7 in the preparation of a detection product for detecting the influenza A virus H5N1 HA protein.

14. Use of the antibody of any one of claims 1-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the host cell of claim 6, the detection reagent of claim 7, and / or the detection product of claim 8 in detecting the influenza A virus H5N1 HA protein for non-diagnostic purposes.

15. Use of the antibody of any one of claims 1-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the host cell of claim 6, the detection reagent of claim 7, and / or the detection product of claim 8 in the preparation of a product for diagnosing influenza A virus H5N1 infection disease.

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