Monoclonal antibody against hemagglutinin protein of avian influenza virus H7 subtype and application thereof

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a monoclonal antibody against hemagglutinin protein of H7 subtype of avian influenza virus and application. The CDR amino acid sequences of the heavy chain variable region of the monoclonal antibody 1F4 are shown as SEQ ID NO. 1-3, the CDR amino acid sequences of the light chain variable region are shown as SEQ ID NO. 4-6, and the heavy chain, the light chain variable region and the coding nucleotide sequence are clear. The monoclonal antibody has high specificity and good affinity, can effectively distinguish the H7 subtype from other influenza virus subtypes, and has no cross reaction. The application also provides application of the monoclonal antibody in preparation of a recognition tool, and the constructed colloidal gold detection test strip takes the 1F4 as a capture and label antibody, is simple and convenient to operate, the result is intuitive, is suitable for on-site rapid screening and large-scale monitoring, and provides technical support for prevention and control of the H7 subtype of avian influenza.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a monoclonal antibody against the H7 subtype hemagglutinin protein of avian influenza virus and its application. Background Technology

[0002] Influenza A viruses are a class of pathogens that have a significant impact on human and animal health, spreading globally for a long time and posing a persistent threat. The viral genome consists of eight independent RNA segments, each encoding one to three proteins, including alkaline polymerase 2 (PB2), alkaline polymerase 1 (PB1), acid polymerase (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix protein (M), and non-structural protein (NS). These proteins play key roles in viral replication, assembly, and pathogenesis.

[0003] Hemagglutinin (HA) is the most important glycoprotein on the surface of influenza viruses, playing a crucial role in viral adsorption and invasion of host cells. Based on the antigenic differences of HA proteins, influenza viruses are classified into different subtypes. Currently, 18 HA subtypes have been identified, with H1-H16 being the main subtypes detected in poultry. These 18 subtypes can be further divided into two major groups: Group 1 (including subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18) and Group 2 (including subtypes H3, H4, H7, H10, H14, and H15). Highly pathogenic avian influenza is mainly caused by specific strains of the H5 and H7 subtypes. The H7 subtype strain spreads rapidly in poultry, has a high mortality rate, and some strains can infect humans across species, with a high mortality rate in severe cases. Due to its rapid spread and serious harm, highly pathogenic avian influenza has been classified as a Category A animal disease by the World Organisation for Animal Health.

[0004] Clinical manifestations of H7 subtype avian influenza virus infection in poultry vary considerably. Mild cases present with respiratory symptoms or decreased egg production, while severe cases can lead to serious systemic lesions and high mortality rates, severely impacting the poultry industry. Some strains (such as H7N9) not only exhibit strong transmissibility and pathogenicity in poultry but can also cross species boundaries to infect humans, causing severe respiratory illnesses. Currently, there are no specific clinical treatments for H7 subtype avian influenza virus, and epidemic prevention and control mainly rely on vaccination, biosafety management, and culling and quarantine measures after an outbreak. However, due to frequent viral mutations, existing vaccines cannot consistently cover the ever-evolving circulating strains, resulting in high control costs and significant losses, placing enormous pressure on long-term prevention and control efforts.

[0005] Against this backdrop, establishing efficient and reliable detection methods is crucial for epidemic prevention and control. Currently, the detection of H7 subtype avian influenza virus mainly relies on laboratory nucleic acid testing and rapid immunological testing. The latter, due to its ease of operation and intuitive results, is more suitable for on-site screening and large-scale monitoring. However, some existing immunological testing products still use polyclonal antibodies as the main recognition element, which are prone to cross-reactivity in practical applications and cannot meet the needs for accurate identification of viral subtypes. Therefore, developing an immunological detection method that can accurately identify H7 subtype avian influenza virus is of great significance for achieving timely detection and precise control of the epidemic, reducing the risk of transmission and economic losses.

[0006] Hemagglutinin (HA) protein is a key antigen determining the subtype specificity of influenza viruses. Its epitopes include both conserved regions and highly specific variant regions. In actual antibody screening, effectively avoiding interference from conserved epitopes during immunization and screening processes to obtain high-affinity monoclonal antibodies against H7 subtype-specific epitopes is a key technical challenge for improving detection specificity. Therefore, developing monoclonal antibodies with high specificity and high affinity against the H7 subtype HA protein is of great significance for constructing high-performance immunoassay systems. Summary of the Invention

[0007] This invention utilizes hybridoma technology to screen and obtain monoclonal antibody 1F4, which specifically recognizes the H7-HA protein. This monoclonal antibody 1F4 can efficiently recognize the H7-HA recombinant protein, exhibiting good specificity and sensitivity. This invention applies the monoclonal antibody to an immunoassay platform, constructing a rapid test strip or test card based on colloidal gold immunochromatography. This test strip exhibits high sensitivity to the H7-HA recombinant protein and shows no cross-reactivity with other proteins, solving the technical problem of cross-reactivity and difficulty in accurately identifying viral subtypes with polyclonal antibodies in existing avian influenza virus H7 subtype detection methods.

[0008] To achieve the above objectives, the main technical solutions adopted by the present invention include: This application provides a monoclonal antibody against the hemagglutinin protein of avian influenza virus H7 subtype. The monoclonal antibody is named monoclonal antibody 1F4. The monoclonal antibody includes a heavy chain variable region and a light chain variable region. The heavy chain variable region of the monoclonal antibody includes three complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.1, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.3. The light chain variable region of the monoclonal antibody includes three complementarity-determining regions, CDR-L1, CDR-L2, and CDR-L3. The amino acid sequence of CDR-L1 is shown in SEQ ID NO.4, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.5, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.6.

[0009] In some embodiments, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.7; The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.8.

[0010] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO. 9.

[0011] In some embodiments, the nucleotide sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO. 10.

[0012] Secondly, the application of the monoclonal antibody provided in this application in the preparation of a tool for recognizing the H7 hemagglutinin protein of avian influenza virus.

[0013] In some embodiments, the tools include reagents, kits, test strips, and antibody chips; The tool is used to identify the H7 hemagglutinin protein of avian influenza virus in samples selected from any of the following: environmental samples, swab samples, cell culture supernatants, and tissue homogenates. The identification is not intended for disease diagnosis.

[0014] Environmental samples refer to non-biological samples collected from poultry activity or processing sites during avian influenza virus surveillance. These mainly include wastewater from poultry washing, swabs from cutting board surfaces, poultry feces, swabs from cages, and swabs from the ground. These samples are usually collected from places such as live poultry markets, poultry farms, slaughterhouses, farmers' markets, and transport vehicles.

[0015] Swab samples refer to samples collected from live poultry, such as the oropharynx, cloaca, nasal cavity, and trachea, using sterile cotton swabs or special sampling swabs in avian influenza virus testing.

[0016] The test strips include colloidal gold test strips.

[0017] In some embodiments, the colloidal gold test strip uses the monoclonal antibody as both a capture antibody and a labeling antibody.

[0018] In some embodiments, the colloidal gold test strip includes a nitrocellulose membrane, a gold label pad, a sample pad, and absorbent paper sequentially connected to a backing plate.

[0019] In some embodiments, the nitrocellulose membrane is provided with a detection line and a control line; the detection line is coated with the monoclonal antibody, the control line is coated with goat anti-mouse IgG, and the gold-labeled pad is coated with the monoclonal antibody.

[0020] Beneficial effects: This invention clarifies the amino acid sequences of the complementarity-determining regions (CDRs) of the heavy chain and light chain variable regions of the monoclonal antibody 1F4, as shown in SEQ ID NO.1–SEQ ID NO.3 and SEQ ID NO.4–SEQ ID NO.6, respectively. The antibody exhibits high specificity and good affinity, effectively distinguishing the H7 subtype from other influenza virus subtypes (such as H1, H3, H5, H9, H10, etc.), and does not cross-react with influenza A NP protein, Newcastle disease virus NP protein, or other common avian virus vaccines. A colloidal gold immunochromatographic test strip constructed based on this monoclonal antibody, using 1F4 as both the capture and labeling antibody, achieves highly sensitive detection of the H7-HA recombinant protein, with a detection limit as low as 1 ng / mL. The test strip has a rational structural design, including a detection line coated with 1F4 and a control line coated with goat anti-mouse IgG. It is easy to operate, provides intuitive results, and exhibits good stability, making it suitable for rapid on-site screening and large-scale monitoring. In addition, this monoclonal antibody can also be applied to the development of various detection tools such as reagents, kits, and antibody chips, providing an efficient and reliable immunological tool for the rapid detection of H7 hemagglutinin protein of avian influenza virus. It is suitable for avian disease monitoring, epidemiological investigation and biosafety control, and provides technical support for the timely detection and effective control of avian influenza epidemics. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The image shows the identification results of the purified monoclonal antibody; Figure 2 This is a schematic diagram of colloidal gold assembly; Figure 3 The results show the specificity of H7-HA monoclonal antibody colloidal gold test strips for multiple avian influenza HA subtypes and other viral recombinant proteins. Figure 4 This refers to the specific detection results of the H7-HA monoclonal antibody colloidal gold test strip for virus or vaccine antigens. Figure 5 This is a graph showing the sensitivity test results of the test strips; Figure 6 This is a graph showing the results of antibody binding identification. Detailed Implementation

[0023] The embodiments of this application will be further described in detail below with reference to the accompanying drawings and examples. The detailed description of the following embodiments and the accompanying drawings are used to illustrate the principles of this application by way of example, but should not be used to limit the scope of this application. This application can be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

[0024] This invention utilizes hybridoma technology to successfully obtain a monoclonal antibody that specifically recognizes the HA protein of the H7 subtype avian influenza virus, and constructs a colloidal gold immunochromatographic test strip based on this antibody. Verification has shown that this monoclonal antibody and its pairing system exhibit good detection sensitivity and specificity against the H7 recombinant protein antigen, meeting the application requirements for rapid on-site screening and providing a raw material basis for the development of diagnostic reagents for the H7 subtype avian influenza virus.

[0025] The detection method described in this application does not involve the diagnosis of diseases.

[0026] H7 hemagglutinin protein, also known as H7-HA protein, includes H7-HA recombinant protein and H7-HA natural protein.

[0027] Example 1 1. Screening of H7-HA monoclonal antibodies 1.1 Mouse Immunization Mice were immunized with H7-HA recombinant protein (A / Shanghai / 1 / 2013 HA, 40104-V08B, Sinocare). The H5 recombinant protein (A / Anhui / 1 / 2005 HA, 11048-V08B, Sinocare), carrying the same His tag, was used as a retroscreening antigen for monoclonal antibody screening. Because different HA subtypes share some spatial homology, conventional screening easily yields antibodies targeting conserved epitopes, leading to cross-reactivity with other subtypes (such as H5). The H7 subtype belongs to Group 2, while the H5 subtype belongs to Group 1; both exhibit significant differences in their antigenic epitopes and are representative. Therefore, introducing the H5 subtype as a retroscreening antigen helps effectively exclude antibodies recognizing conserved HA structural regions, preferentially screening for monoclonal antibodies targeting H7 subtype-specific epitopes, thereby improving antibody specificity and detection accuracy.

[0028] Specifically, purified H7-HA recombinant protein was mixed with an equal volume of Freund's complete adjuvant (total volume 200 μL) and subcutaneously injected at multiple sites into 6-week-old female BALB / c mice at a dose of 30 μg / mouse. In weeks 2 and 4, booster immunizations were administered subcutaneously at multiple sites with the same dose mixed with an equal volume of Freund's incomplete adjuvant. In week 6, the spleen of mice was directly injected with insulin at a dose of 10 μg / mouse. Seven days after the final immunization, mouse serum was collected to detect antibody titers. Mice with high titers were selected for a booster immunization of 30 μg H7-HA recombinant protein via intraperitoneal pulse, and the spleen was collected 3 days later for hybridoma cell preparation.

[0029] 1.2 Screening of hybridoma cells All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in logarithmic growth phase and then cultured in HAT medium for selection. When the fused cells reached halfway up the bottom of the well, clones positive for both H7-HA recombinant protein were obtained by indirect ELISA. Since the immunogen contained a His tag, background components needed to be screened to identify specific cell lines targeting H7-HA recombinant protein. Positive cells were cloned to monoclonal status using limiting dilution, and then the cell lines were expanded and cryopreserved.

[0030] 1.3 Screening of positive clones using indirect ELISA method Recombinant H7-HA protein and other His-tagged recombinant proteins (H5) were coated in microplates (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water) at a concentration of 1 μg / mL, and incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 150 μL of 3% sucrose + 2% BSA per well, and incubated at 37°C for 2 hours. The plates were then washed once with PBST (PBS containing 0.05% Tween-20, pH 7.4) and blotted dry. 50 μL of cell culture supernatant was added, and the plates were incubated at 37°C for 30 min. Discard the liquid from the wells, wash the plate four times with PBST, blot dry, and add 50 μL / well of HRP-labeled goat anti-mouse IgG (Solepro, diluted 5000 times with PBS). Incubate at 37°C for 30 min, wash four more times, blot dry, and add 50 μL / well of TMB chromogenic buffer for incubation at room temperature for 10 min. Finally, add 50 μL of TMB stop solution (Beijing Meikewande, 1001SA) to stop the reaction. Measure the OD using a microplate reader. 450 nm value. Positive cell lines that reacted with the H7-HA recombinant protein but not with the control recombinant protein were selected for subsequent experiments.

[0031] Table 1: Screening results of monoclonal antibodies

[0032] After the selected hybridoma cell lines were expanded and cultured, 0.2 ml (containing 2.5 × 10⁻⁶ cells) was injected intraperitoneally. 6 Female BALB / c mice (cells) were used to collect ascites fluid approximately 10 days later, when the mice’s abdomens were noticeably swollen.

[0033] 2. Purification and Identification of Monoclonal Antibodies Centrifuge the ascites fluid at 12000 rpm for 10 minutes, collect 1 ml of the supernatant, dilute it 10-fold with binding buffer (20 mM PB, 150 mM NaCl, pH 7.4), and filter it through a 0.22 μm filter. Pump the filtered sample slowly into a Protein L purification column equilibrated with binding buffer using a peristaltic pump. Connect the column to a protein purification instrument and wash with binding buffer for 5-10 column volumes until the UV absorption peak flattens. Then elute with elution buffer (0.1 M glycine, pH 2.7), collect the elution peak, and adjust the collected sample to neutral with 1 M Tris-HCl (pH 9). Transfer the solution to a dialysis bag (MW: 8000-14000) and dialyze in 20 mM PBS (pH 7.4) at 2-8°C for 16 hours. Transfer the liquid from the dialysis bag to a centrifuge tube and centrifuge at 12000 rpm for 5 minutes. The supernatant is the purified monoclonal antibody.

[0034] The purified monoclonal antibody was diluted 1 μg / ml, and its binding activity with H7-HA recombinant protein and control antigen H5 recombinant protein was detected by indirect ELISA. See below for specific results. Figure 1 .according to Figure 1 It can be seen that the selected monoclonal antibody specifically binds to the H7-HA protein and does not react with the control antigen H5, indicating that the monoclonal antibody has good specificity and can be used for subsequent testing.

[0035] 3. Colloidal gold pairing of H7-HA monoclonal antibodies Preparation of antibody-colloidal gold labeled complex.

[0036] Antibody labeling: Colloidal gold solution was prepared using the trisodium citrate reduction method. The specific procedure was as follows: 100 mL of 0.01% chloroauric acid solution was heated to boiling, and then 1 mL of 1% trisodium citrate solution was quickly added until the solution turned wine-red. Boiling was continued for 5 minutes, and the colloidal gold particles were allowed to stabilize before cooling to room temperature. 1 mL of colloidal gold solution was placed in a centrifuge tube, and 0.2 M potassium carbonate solution was added in gradients of 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, and 6 μL to obtain the optimal pH for efficient antibody-colloidal gold conjugation. The optimal conjugation effect was found to be 5 μL. After mixing, 5 μg of the H7-HA monoclonal antibody to be labeled was added, and the mixture was quickly mixed and incubated at room temperature for 10 min. Then, 10 μL of 10% (w / v) bovine serum albumin (BSA) was added to block non-specific binding sites, and the mixture was incubated at room temperature for another 10 min. Add 10 μL of 10% (w / v) polyethylene glycol 20000 (PEG20000) to enhance labeling stability. After mixing, centrifuge at 12000 rpm for 10 min and discard the supernatant. Resuspend the lower precipitate in 1 / 10 volume of reconstitution solution (0.05M Tris + 0.1% N100 + 1% Sucrose + 1% BSA + 0.1% PC300, pH 8.6) to obtain the antibody-colloidal gold labeled complex. Store at 4°C protected from light for later use.

[0037] 4. Preparation of test strips coated with different monoclonal antibodies against H7-HA The selected H7-HA monoclonal antibodies were scribed onto nitrocellulose membranes of different sizes (20mm × 300mm). Diluted monoclonal antibodies (antibody dilution solution: 0.01M PB + 0.5% BSA + 0.5% Trehalose, pH 7.6) were horizontally scribed in a linear fashion on each membrane, with a scribing volume of 0.8 μL / cm, forming the detection line (T line). Goat anti-mouse IgG antibody, diluted in 0.01M PBS at pH 7.4 to a concentration of 1 mg / mL, was then scribed horizontally in a linear fashion at a volume of 0.8 μL / cm onto the nitrocellulose membrane, forming the control line (C line).

[0038] 5. Screening of paired monoclonal antibodies Nitrocellulose membranes streaked with different monoclonal antibodies against H7-HA were individually paired with different colloidal gold-labeled monoclonal antibodies. H7-HA protein was diluted to 20 ng / mL for detection, and H5 recombinant protein was also diluted to 20 ng / mL as a negative antigen for detection. Combinations that showed the strongest color development for H7-HA protein and did not react with the control H5 protein were screened. The screening results are shown in Table 2. Therefore, the optimal pairing for detecting H7-HA recombinant protein was determined to be 1F4 streaking and 1F4 gold labeling.

[0039] Table 2: Results of screening paired monoclonal antibodies using H7-HA recombinant protein

[0040] - indicates a negative result, meaning no color develops; + / ++ / +++ indicates a positive result, meaning a color reaction occurs. The more + signs there are, the deeper the color, and the stronger the positive reaction.

[0041] Table 2 shows the screening results using H7-HA recombinant protein diluted to a concentration of 20 ng / mL as a positive antigen. The results for negative antigen and blank dilution were all negative and are not shown in Table 2. The results showed that the combination of 1F4 monoclonal antibody streaking and 1F4 monoclonal antibody labeled with gold produced the deepest staining of H7-HA recombinant protein, making it the optimal pairing. That is, the combination of monoclonal antibody anti-1F4 as the capture antibody and monoclonal antibody 1F4 as the labeling antibody can specifically recognize H7-HA recombinant protein.

[0042] 6. Preparation and assembly of colloidal gold test strips Preparation of gold-labeled pads: Using a 6mm×300mm glass fiber membrane, the prepared colloidal gold-labeled antibody was evenly dropped onto the glass fiber at a rate of 1200μL / strip, allowed to air dry naturally, and then dried at 37℃ for 2 hours for later use.

[0043] See Figure 2 , Figure 2 This is a schematic diagram of the colloidal gold assembly. A 60mm × 300mm PVC backing plate is used as a support. Sample pads, gold-labeled pads (also called gold-labeled binding pads), nitrocellulose membranes, and absorbent paper are attached to the backing plate. The nitrocellulose membrane is coated with two lines and dried at 37℃ for 12 hours before use. The nitrocellulose membrane is coated with a detection line (monoclonal antibody 1F4 for streaking) and a control line (goat anti-mouse IgG). The gold-labeled pad is coated with monoclonal antibody 1F4. The assembled plate is cut into 4mm strips using a strip cutter and wrapped with colloidal gold plastic casings. The sample pads are exposed at the sample application wells of the plastic casings, while the control and detection lines are exposed at the result observation wells. The colloidal gold test strip is now assembled.

[0044] 7. Test strip specificity test Recombinant protein samples: H1 recombinant protein (A / California / 04 / 2009 HA), H3 recombinant protein (A / Perth / 6 / 2009 HA, 40035-V08H, Sinocare), H5 recombinant protein (A / Anhui / 1 / 2005 HA, 11048-V08B, Sinocare), H7 recombinant protein, H9 recombinant protein (A / chicken / Hong Kong / G9 / 1997 HA, 40036-V08H, Sinocare), H10 recombinant protein (A / Jiangxi-Donghu / 346 / 2013 HA, 40359-V08B, Sinocare), influenza A virus NP protein (expressed by the inventor using E. coli, FluA-NP), and Newcastle disease virus NP protein (expressed by the inventor using E. coli, NDV-NP) were diluted to 100 ng / mL with sample diluent for detection.

[0045] The amino acid sequence of the NP protein of influenza A virus is shown in SEQ ID NO.11: MASQGTKRSYEQMETGGERQDATEIRASVGRMIGGIGRFYIQMCTELKLSDYDGRLIQNSITIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRIDGKWMRELILYDKEEIRRVWRQAN NGEDATAGLTHIMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTIAMELIRMIKRGINDRNFWRGENGRRTRVAYERMCNILKGKFQTAAQRAMMDQVRESRNPG NAEIEDLIFLARSALILRGSVAHKSCLPACVYGLAVASGHDFEREGYSLVGIDPFKLLQNSQVVSLMRPNENPAHKSQLVWMACHSAAFEDLRVSSFIRGKKVIPRGKLSTRGVQIASNENVETM DSNTLELRSRYWAIRTRSGGNTNQQKASAGQISVQPTFSVQRNLPFERATVMAAFSGNNEGRTTSDMRTEVIRMMESAKPEDLSFQGRGVFELSDEKATNPIVPSFDMSNEGSYFFGDNAEEYDS.

[0046] The NP protein nucleotides of influenza A virus are shown in SEQ ID NO.12:

[0047] The amino acid profile of the Newcastle disease virus NP protein is shown in SEQ ID NO. 13: MSSVFDEYEQLLASQTRPNGSHGGGEKGSTLKVEVPVFTLNSDDPEDRWNFAVFCLRIAVSEDANKPLRQGALISLLCTHSQVMRNHVALAGRQNEATLAILEIDGFSNGVPQFNNRSGVSE ERAQRFMMIAGSLPRACSNGTPFVTAGVEDDAPEDITTDTLERILSIQVQVWVTVAKAMTAYETADESETRRINKYMQQGRVQKRCILHPVCRSAIQLTIRQSLAVRIFLVSELKRGRNTAGGT STYYNLVGDVDSYIRNTGLTAFFLTLKYGINTKTSVLALSSLSGDIQKMKQLMRLYRMKGENAPYMTLLGDSDQMSFAPAEYAQLYSFAMGMASVLDKGTVKYQFARDFMSTSFWRLGVEYA QAQGSSINEDMAAELKLTPAVRRGLAAAAQRVSEDASNMDLPTQQAGVLTGLSDNTPPAQPGGSKPQGSADGNEGETQFLDLMRAVANSMRDAPNSAQGSSQPAPPPTPGGNQDNDTDWGY.

[0048] The NP protein nucleotides of Newcastle disease virus are shown in SEQ ID NO. 14:

[0049] Virus or vaccine antigen samples: inactivated PR8 mouse lung-adapted strain (H1N1), avian influenza H9 subtype inactivated vaccine (NJ01 strain, Shandong Huahong), Newcastle disease virus (NDV) live vaccine (LaSota strain, Qingdao Yibang), and infectious bronchitis (IBV) live vaccine (H120 strain, Qingdao Yibang). The vaccines were reconstituted according to the instructions and then diluted 10 times with the sample diluent before testing.

[0050] Add 80 μL of the diluted sample to the sample well of the test strip. Simultaneously, add another 80 μL of the diluent to a new test strip as a blank control. Determine the results within 20 minutes. If both the T and C lines show clear red bands, the result is positive; if only the C line shows color, the result is negative; if the C line does not show color, the result is invalid.

[0051] Figure 3 , Figure 4 As shown, the test strip can produce a clear detection signal for H7-HA recombinant protein, but does not react with other HA subtype recombinant proteins such as H1, H3, H5, H9, and H10, as well as influenza A virus NP protein and Newcastle disease virus NP protein. It also shows no cross-reaction with PR8 mouse lung-adapted strain, avian influenza H9 vaccine, Newcastle disease virus vaccine, and infectious bronchitis virus vaccine, indicating that the test strip has good specificity.

[0052] 8. Sensitivity test of test strips The H7-HA recombinant protein was diluted at concentrations of 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.5 ng / mL, and 0.25 ng / mL before detection. Figure 5 The results showed that the colloidal gold test strip still showed weak color development at a recombinant protein concentration of 1 ng / mL, while the blank dilution, i.e., the sample dilution (0.01M Tris + 0.15M NaCl + 2% Sucrose + 0.5% BSA + 0.15% skim milk powder + 0.1% Tween-20 + 0.1% N100 + 0.01% PC300, pH 8.0) (0 ng / mL), did not show color development, indicating that the limit of detection for H7-HA recombinant protein on the test strip is 1 ng / mL.

[0053] 9. Monoclonal antibody binding activity assay Based on the selection of potential paired antibodies using colloidal gold, the selected paired monoclonal antibody 1F4 and other unrelated murine monoclonal antibodies were serially diluted (to concentrations of 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, and 100 pg / mL, respectively) using the aforementioned indirect ELISA method to evaluate their binding activity with the H7-HA recombinant protein. Influenza A virus NP murine monoclonal antibody was used as a negative control to exclude the influence of non-specific binding. Results are shown below. Figure 6 . Figure 6 In the diagram, "Ctrl" represents the negative control, influenza A virus NP mouse monoclonal antibody 1B5 (inventor's commercially available product, M100014). Monoclonal antibody 1F4 showed a significant positive signal with the H7 protein at a concentration of 10 ng / mL, indicating strong binding activity.

[0054] 10. Gene sequence of monoclonal antibodies Total RNA was extracted from hybridoma cells using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using RandomPrimers. Universal primers for the variable region of mouse antibodies were designed, and the VH and VL genes were amplified by PCR in three rounds. The PCR products were purified by gel excision and ligated into the pUC19 vector, transformed into TOP10 strain, and single colonies were picked and sequenced after culturing at 37°C for 14 h to obtain the gene sequences of the light and heavy chains of the monoclonal antibody.

[0055] The sequence of the gold monoclonal antibody 1F4 was obtained by stenography / labeling.

[0056] Light chain variable region nucleotide sequence: The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1F4 is shown in SEQ ID NO.10: GAAATTGTGCTCACTCAGTCTCCATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTATTTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTATTAAACTCCTGATCTACTACACATCAAGATTACAC TCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAACAAGAAGATATTGCCACTTACTTTTGTCAACAGGGTTATTCTCTTCCTCCGACGTTCGGTGGCGGCACCAAGCTGGAAATCAAACGTACGGTG.

[0057] Light chain variable region amino acid sequence: The amino acid sequence of the light chain variable region of the monoclonal antibody 1F4 is shown in SEQ ID NO. 8: EIVLTQSPSSLSASLGDRVTISCRASQDISIYLNWYQQKPDGTIKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGYSLPPTFGGGTKLEIKRTV.

[0058] Light chain CDR area annotation: The amino acid sequence of the light chain variable region CDR-L1 of monoclonal antibody 1F4 is shown in SEQ ID NO.4: CDR-L1: RASQDISIYLN; The amino acid sequence of the light chain variable region CDR-L2 of monoclonal antibody 1F4 is shown in SEQ ID NO. 5: CDR-L2: YTSRLHS; The amino acid sequence of the light chain variable region CDR-L3 of monoclonal antibody 1F4 is shown in SEQ ID NO. 6: CDR-L3: QQGYSLPPT.

[0059] Heavy chain variable region nucleotide sequence: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1F4 is shown in SEQ ID NO.9: GAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTACTTATGGCATGTCTTGGGTTCGCCAGACTCCAGACAAGAGGCTGGAGTTGGTCGCAACCATTAATAATAATGGTGATAAG ATCTTTTTATCCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAAAACATCCTGTACCTGCAAGTGAGCAGTCTGAGGTCTGAAGACACAGCCATTTATTGTTTAAGAGGCTCCTTTGACTACTGGGGCCAAGGCACCACTCTCACCGTCTCCTCA.

[0060] Heavy chain variable region amino acid sequence: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1F4 is shown in SEQ ID NO.7: EVQLQESGGGLVQPGGSLKLSCAASGFTFSTYGMSWVRQTPDKRLELVATINNNGDKIFYPDSVKGRFTISSRDNAKNILYLQVSSLRSEDTAIYYCLRGSFDYWGQGTTLTVSS.

[0061] Heavy chain CDR region annotation: The amino acid sequence of the heavy chain variable region CDR-H1 of the monoclonal antibody 1F4 is shown in SEQ ID NO.1: CDR-H1: TYGMS; The amino acid sequence of the heavy chain variable region CDR-H2 of the monoclonal antibody 1F4 is shown in SEQ ID NO.2: CDR-H2: TINNNGDKIFYPDSVKG; The amino acid sequence of the heavy chain variable region CDR-H3 of the monoclonal antibody 1F4 is shown in SEQ ID NO.3: CDR-H3: GSFDY.

Claims

1. A monoclonal antibody against the hemagglutinin protein of avian influenza virus H7 subtype, characterized in that, The monoclonal antibody includes a heavy chain variable region and a light chain variable region. The heavy chain variable region of the monoclonal antibody includes three complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.1, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.

3. The light chain variable region of the monoclonal antibody includes three complementarity-determining regions, CDR-L1, CDR-L2, and CDR-L3. The amino acid sequence of CDR-L1 is shown in SEQ ID NO.4, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.5, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

6.

2. The monoclonal antibody against avian influenza virus H7 subtype hemagglutinin protein according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.7; The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

8.

3. The monoclonal antibody against avian influenza virus H7 subtype hemagglutinin protein according to claim 2, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.

9.

4. The monoclonal antibody against avian influenza virus H7 subtype hemagglutinin protein according to claim 2, characterized in that, The nucleotide sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

10.

5. The use of the monoclonal antibody according to claim 1 in the preparation of a tool for recognizing the H7 hemagglutinin protein of avian influenza virus.

6. The application according to claim 5, characterized in that, The tools include reagents, kits, test strips, and antibody chips; The test strips include colloidal gold test strips.

7. The application according to claim 6, characterized in that, The colloidal gold test strip uses the monoclonal antibody as both the capture antibody and the labeling antibody.

8. The application according to claim 7, characterized in that, The colloidal gold test strip comprises a nitrocellulose membrane, a gold label pad, a sample pad, and absorbent paper, which are sequentially connected to a back plate.

9. The application according to claim 8, characterized in that, The nitrocellulose membrane is provided with a detection line and a control line; the detection line is coated with the monoclonal antibody, the control line is coated with goat anti-mouse IgG, and the gold label pad is coated with the monoclonal antibody.

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