H9n2 subtype avian influenza virus ha1 protein monoclonal antibody and preparation method and application thereof

Abstract

The application provides a H9N2 subtype avian influenza virus HA1 protein monoclonal antibody and a preparation method and application thereof, and belongs to the technical field of monoclonal antibodies.The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.2, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4, the antibody titer is as high as 1:2.04x10 6 , the affinity constant is 1.103x10 9 L / mol, the specificity is strong, the monoclonal antibody can be specifically combined with the H9N2 subtype avian influenza virus HA1 protein, and there is no cross reaction.The monoclonal antibody can be used for detection and diagnosis of the H9N2 subtype avian influenza virus, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of monoclonal antibody technology, and in particular to a monoclonal antibody against the HA1 protein of H9N2 subtype avian influenza virus, its preparation method, and its application. Background Technology

[0002] Avian influenza virus (AIV) belongs to the genus *Influenza A* of the family Orthomyxoviridae. It is an important zoonotic pathogen, with poultry as its natural host and the core vector for the storage, transmission, and mutation of this virus. The clinical symptoms of poultry infected with AIV are typical and specific, mainly manifested as sudden onset, a rapid rise in body temperature, accompanied by lethargy, such as standing still, closing eyes, and drowsiness; significant respiratory symptoms, including severe difficulty breathing, mucus discharge from the mouth, often accompanied by frequent head shaking, swallowing movements, and hoarseness; significant impact on the reproductive system, with a sudden and sharp drop or even complete cessation of egg production; and gastrointestinal symptoms, including the excretion of yellowish-white, yellowish-green, or green watery feces, which can lead to death in severe cases.

[0003] Among the many AIV subtypes, the H9N2 subtype is the most widespread and has the highest infection rate in chicken flocks in my country. It is also one of the most prevalent and destructive avian influenza virus subtypes globally. It is noteworthy that although the mortality rate of H9N2 subtype avian influenza virus when infecting poultry alone is relatively low, it is highly susceptible to co-infection with other pathogens, significantly increasing morbidity and mortality rates in poultry and causing huge economic losses to the poultry farming industry. More critically, H9N2 subtype AIV can be transmitted to mammals, including humans, through direct or indirect contact with poultry, posing a zoonotic risk. This not only seriously threatens the healthy and sustainable development of the poultry industry but also presents a potential and ongoing challenge to global public health security.

[0004] Monoclonal antibodies, with their unique advantages such as high specificity (precise recognition of a single antigenic epitope), high batch-to-batch stability, and scalable industrial production, have become a core tool in the development of biological detection and diagnostic reagents, playing an irreplaceable role in precise pathogen detection and disease control. Since Kohler and Milstein established the B lymphocyte hybridoma technology in 1975, the large-scale preparation technology of murine monoclonal antibodies has made breakthrough progress, laying a mature technical foundation for the development of highly specific pathogen detection reagents and promoting the rapid development of the field of biological diagnostics.

[0005] However, to date, the development of highly specific murine monoclonal antibodies targeting the active sites (such as key cysteine ​​residue regions) or unique conformational epitopes of the HA1 protein of the H9N2 subtype avian influenza virus still has shortcomings. Their practical application technology in the accurate detection of H9N2 AIV (such as the development of specific diagnostic kits) is not yet fully mature, failing to meet the current demand for rapid and accurate detection of H9N2 subtype AIV in the poultry industry, and also unable to effectively support the prevention and control of this virus. Therefore, developing highly specific monoclonal antibodies targeting H9N2 AIV to fill the gaps in related detection technologies is of great significance for improving the level of H9N2 AIV prevention and control, ensuring the safety of the poultry industry and global public health security, and is also a key technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] In view of this, the present invention provides a monoclonal antibody against the HA1 protein of H9N2 subtype avian influenza virus, its preparation method and application, in order to solve the above problems.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a monoclonal antibody against the HA1 protein of H9N2 subtype avian influenza virus. The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4.

[0009] The present invention also provides a nucleic acid molecule encoding the monoclonal antibody, the nucleic acid molecule comprising nucleotide sequences encoding a heavy chain variable region and a light chain variable region; the sequence encoding the heavy chain variable region is shown in SEQ ID NO.1, and the sequence encoding the light chain variable region is shown in SEQ ID NO.3.

[0010] The present invention also provides a method for preparing the monoclonal antibody, comprising the following steps:

[0011] S1. Immunize BALB / c mice with HA1 protein, and then fuse spleen cells of the immunized mice with SP2 / 0 myeloma cells to obtain hybridoma cells;

[0012] S2. Using HA1 protein as the coating antigen, positive hybridoma cells were screened by indirect ELISA, and hybridoma cell lines that stably secrete monoclonal antibodies were obtained by subcloning.

[0013] S3. The hybridoma cell line was inoculated into the peritoneal cavity of pretreated mice to induce ascites and purify it to obtain the monoclonal antibody.

[0014] Preferably, the HA1 protein is the protein encoded by the HA1 gene after codon optimization.

[0015] Preferably, the immunization process in step S1 is as follows: HA1 protein is emulsified with Freund's complete adjuvant for primary immunization, and booster immunization is performed every 2 weeks using the same method and dosage as the primary immunization, for a total of 3 booster immunizations; super-strong immunization is performed 3-5 days before cell fusion.

[0016] Preferably, the dose of HA1 protein for primary and booster immunizations is 48–52 μg independently.

[0017] Preferably, the super-immune response is achieved by injecting BALB / c mice with HA1 protein without adjuvant, with an immunization dose of 18-22 μg per mouse.

[0018] Preferably, HA1 protein and Freund's complete adjuvant are emulsified after being mixed at a volume ratio of 0.8~1.2:0.8~1.2.

[0019] Preferably, in step S3, Protein A affinity chromatography is used for purification, with PBS solution as the binding buffer, 0.1M glycine as the elution buffer, and 1M Tris-HCl as the neutralization buffer.

[0020] The present invention also provides the application of the monoclonal antibody in the preparation of a detection kit or reagent kit for H9N2 subtype avian influenza virus.

[0021] By adopting the above technical solution, the present invention has the following beneficial effects: the amino acid sequence of the heavy chain variable region of the monoclonal antibody of the present invention is shown in SEQ ID NO.2, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4, and the antibody titer is as high as 1:2.04×10⁻⁶. 6 The affinity constant is 1.103 × 10⁻⁶. 9 The antibody has a concentration of L / mol and high specificity, binding specifically to the HA1 protein of the H9N2 subtype avian influenza virus without cross-reactivity. This monoclonal antibody can be used for the detection and diagnosis of the H9N2 subtype avian influenza virus and has promising application prospects. Attached Figure Description

[0022] Figure 1 The results are SDS-PAGE assays for HA1 protein purification; lane M represents the pre-stained protein molecular weight standard; lane 1 represents samples purified by affinity chromatography.

[0023] Figure 2 The results are Western blot identification results of monoclonal antibodies; lane M is the molecular weight standard of pre-stained protein; lane 1 is the purified H9N2 AIV HA1 protein.

[0024] Figure 3The results are SDS-PAGE assays of purified monoclonal antibodies; lane M represents the molecular weight standard of pre-stained proteins; lane 1 represents the unpurified A10 monoclonal antibody sample; and lane 2 represents the A10 monoclonal antibody sample purified by affinity chromatography.

[0025] Figure 4 This is a graph showing the titer of a monoclonal antibody.

[0026] Figure 5 This is a graph showing the affinity assay for monoclonal antibodies.

[0027] Figure 6 This is a graph showing the results of a monoclonal antibody specificity test. Detailed Implementation

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1. Immunogen preparation and immunization

[0030] 1. Preparation of recombinant plasmids

[0031] By comparing homologous sequences of the H9N2 subtype avian influenza virus published in the GenBank database, the genetic evolutionary relationship, conserved gene regions, and antigenic variation characteristics of the strains were analyzed. Based on the screening criteria of clearly defined subtype, complete and reliable sequence annotation, strong representativeness of avian epidemic strains, and high homology with currently circulating strains, A / chicken / Fujian / SW / 2022 (H9N2) (GenBank accession number: PP474085.1) was selected as the research strain. To achieve efficient and soluble expression of the HA1 protein in the eukaryotic 293F system, the HA1 protein sequence was analyzed using GenScript's online codon optimization tool, GenSmart. TM Codon Optimization optimizes codon preferences, as follows:

[0032] Primer pairs

[0033] F:5'-CCGCTCGAGGCCACCATGGAAACCCCAGCGCAGCTTCTCTTCCTC-3' (SEQ ID NO. 18);

[0034] R: 5'-GGAAGATCTTTAACGGCTGCTCCGGGACGGGAC-3' (SEQ ID NO.19) was used to amplify the H9N2HA1 gene. A linearized vector was obtained using the pCAGGS vector with BgIII and XhoI double enzymes. The H9N2HA1 gene and the BgIII and XhoI double digestion products of the pCAGGS vector were added to the reaction system at a molar ratio of 6:1 and ligated using T4 DNA ligase. The components were added sequentially according to Table 1 and mixed thoroughly. Ligation was carried out overnight at 16°C to allow directional cloning into the pCAGGS eukaryotic expression vector, constructing the recombinant plasmid pCAGGS-HA1. The verified recombinant plasmid was transfected into 293F cells, and the cell suspension was collected after 96 h of continuous culture for later use.

[0035] Table 1 Connection System

[0036] pCAGGS vector double enzyme digestion product 1.56 Double enzyme digestion of target gene product 4.26 T4 DNA Ligase 2 10×T4 DNA Ligase Buffer 2

[0037] 2. Expression and purification of HA1 protein

[0038] (1) Large-scale cell culture and transfection

[0039] 293F recombinant cells were seeded into 50 mL shake flasks and cultured at 37°C and 120 rpm. Cell growth density was assessed by cell counting. When the cell culture density reached approximately 1 × 10⁻⁶ cells / mL... 6 Cells were expanded from 50 mL shake flasks to 500 mL shake flasks for high-density culture, and cell counts were performed daily. When the cell density was approximately 2 × 10⁶ cells / mL, the cells were cultured at a density of approximately 10⁶ cells / mL. 6 At cell / mL concentration, the culture medium, recombinant plasmid, and PEI transfection reagent were added to a shake flask at a volume ratio of 50:1:3. After gentle mixing, the mixture was further cultured with shaking. PEI dry powder was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd. After precise weighing, it was fully dissolved in sterile ultrapure water to a concentration of 1 g / L. After pH correction and sterilization by filtration through a 0.22 μm sterile filter membrane, it was aliquoted in the dark and stored at -20℃ for later use.

[0040] (2) Collection and pretreatment of culture supernatant

[0041] After 96 h of transfection culture, the culture supernatant expressing HA1 protein was collected and centrifuged at 12000 r / min, 4℃ for 10 min to remove cell debris and impurities. The supernatant was then filtered through a 0.45 μm filter membrane for further purification. After filtration, the supernatant was stored in an ice box at low temperature to prevent protein degradation.

[0042] (3) Protein purification column pretreatment

[0043] Fix the installed protein purification column, use a pipette to slowly add 2 mL of nickel material to the installed protein purification column, and after the packing material settles naturally, carefully place the upper gasket to fix the packing material; open the control valve to allow 20% (v / v) ethanol in the column to flow out naturally, and then rinse the column with 10 mL of deionized water to completely remove the residual ethanol in the column, thus completing the pretreatment of the purification column.

[0044] (4) Purification column equilibration

[0045] Adjust the control valve of the protein purification device to stabilize the column flow rate. After the flow rate is adjusted, rinse the purification column with at least 10 mL of equilibration buffer at a constant flow rate of 1 mL / min to ensure column bed equilibration and prepare for subsequent protein binding.

[0046] (5) Sample loading and binding

[0047] Add 5 mL of the filtered cell culture supernatant to the equilibrated purification column in batches, adjusting the control valve to ensure the column flow rate does not exceed 1 mL / min, guaranteeing sufficient binding of HA1 protein to the nickel affinity packing material. Quickly collect each batch of flushing fluid. Repeat the loading 2-3 times to improve protein binding efficiency. Collect the last flushing fluid and store it at -20°C for later use.

[0048] (6) Elution of extraneous proteins

[0049] After loading the sample, continuously rinse the purification column with equilibration buffer until there is no obvious protein signal in the effluent (which can be preliminarily judged by a protein indicator) to completely remove unbound contaminating proteins from the column and avoid interfering with the purification of the target protein.

[0050] (7) Elution and identification of target protein

[0051] Elution buffers with concentrations of 20 mM, 50 mM, 80 mM, 100 mM, 150 mM, and 200 mM imidazole were prepared, and gradient elution was used to elute the HA1 recombinant protein bound to the column. 10 μL of each gradient eluent was mixed with 30 μL of G250 protein indicator, and the presence of the target protein was preliminarily identified by observing color changes. The eluent fraction with the darkest color was collected and labeled. During elution, the control valve was adjusted to maintain a flow rate of approximately three drops per second to improve the recovery rate of the target protein. The eluent was aliquoted into 1.5 mL EP tubes (1 mL per tube), and the protein concentration of each tube was measured using a microplate reader. Detailed records were kept, and the resulting product was the purified HA1 protein, which was stored at -20°C for later use.

[0052] (8) Cleaning and preservation of purification column

[0053] After protein purification, the purification column was rinsed with 20 mL of deionized water (ddH2O) to remove residual buffer and protein. Then, the column was rinsed with 1 mol / L NaOH solution to remove any difficult-to-remove impurities. The column was then rinsed again with deionized water until no NaOH residue remained. Finally, the purification column was placed in 20% (v / v) ethanol and stored in a sealed container at 4°C for future reuse.

[0054] (9) Identification of purified proteins

[0055] Add 5× Loading Buffer to the purified HA1 protein eluent at a volume ratio of 4:1, mix thoroughly, and boil for 10 min to denature the protein. Analyze the protein purification effect using 12% SDS-PAGE gel electrophoresis. Figure 1 Simultaneously, using H9N2 AIV-positive serum, the purified HA1 protein was specifically identified by Western blot assay. Figure 2 The results showed that a clear, single protein band appeared at the position corresponding to 55~70 kDa, without obvious tailing, indicating that the protein expression was complete and confirming that the expressed protein was the target HA1 protein.

[0056] Example 2. Preparation of monoclonal antibodies

[0057] 1. Animal immunization

[0058] The HA1 protein purified in Example 1 was used as an immunogen to immunize female BALB / c mice. The specific steps are as follows:

[0059] (1) Freund's complete adjuvant was added to the immunogen HA1 protein at a volume ratio of 1:1, and after emulsification, it was used for the first immunization;

[0060] (2) Two 6-week-old female BALB / c mice were selected and immunized by subcutaneous injection at multiple points on the back. The immunization dose for each mouse was 50 μg.

[0061] (3) After the first immunization, every two weeks, the HA1 protein was mixed with Freund's incomplete adjuvant and emulsified. The mice were then given booster immunizations using the same injection method and immunization dose as in step (2). A total of three booster immunizations were administered.

[0062] (4) After the third booster immunization, blood was collected from the tail vein and the serum specific antibody titer against HA1 protein was determined by conventional methods. Mice with high antibody titers were selected and injected with adjuvant-free HA1 protein 3-4 days before cell fusion for super-strong immunization. The immunization dose for each mouse was 20 μg.

[0063] 2. Cell fusion and monoclonal antibody preparation

[0064] Cell fusion was performed using polyethylene glycol (PEG)-mediated fusion, with the following specific steps: Mice subjected to hyperimmunization in step 1 were sacrificed, and their spleen cells were aseptically harvested and mixed with mouse myeloma cells SP2 / 0 at a cell ratio of 8:1. PEG was then added for cell fusion. The fused cells were seeded into a culture system containing HAT selective medium for selective screening. On day 12 post-fusion, positive hybridoma cells were initially screened using HA1 protein as the coating antigen via indirect ELISA. The specific steps of the indirect ELISA method are as follows:

[0065] (1) Dilute the purified HA1 protein with CBS solution to a coating solution with a concentration of 2 μg / mL and coat the microplate with 100 μL per well and incubate at 37℃ for 2 h.

[0066] (2) Discard the coating solution, wash the microplate with PBST washing solution and pat dry, add 5% (w / v) skim milk to the microplate for blocking, 200 μL per well, and incubate at 37°C for 2 h;

[0067] (3) Discard the blocking solution, wash the ELISA plate with PBST washing solution and pat dry. Add the hybridoma cell supernatant (primary antibody) to the ELISA plate, 100 μL per well. At the same time, set H9N2 AIV positive serum as a positive control and incubate at 37°C for 30 min.

[0068] (4) Discard the primary antibody, wash the ELISA plate thoroughly with PBST washing solution, and then pat it dry;

[0069] (5) Add 100 μL of HRP-labeled goat anti-mouse IgG (secondary antibody) diluted to an appropriate concentration to each reaction well and incubate at 37°C for 30 min.

[0070] (6) Discard the secondary antibody, rinse the ELISA plate thoroughly with PBST washing solution, and then pat dry;

[0071] (7) Add 100 μL of freshly prepared TMB colorimetric solution to each well and react in a dark room for 5 min; then add 100 μL of 2M H2SO4 solution to each well to terminate the colorimetric reaction.

[0072] (8) Read the OD of each well using an ELISA reader. 450 value.

[0073] 3. Subcloning of hybridoma cells

[0074] Subcloning of hybridoma cells was performed using the limiting dilution method: Positive hybridoma cells were diluted with 1640 complete medium to approximately 10 cells / mL. 100 μL of the diluted cell suspension was added to a 96-well cell culture plate pre-coated with 100 μL of feeder cells. The plate was incubated at 37°C with 5% CO2 for 6–8 days. After incubation, positive hybridoma cells were screened again using the indirect ELISA method described above. The subcloning and screening steps were repeated 2–3 times until a hybridoma cell line stably secreting anti-HA1 monoclonal antibody was obtained, named 1A10. The selected positive hybridoma cell line was then expanded through culture at a rate of 1–2 × 10⁻⁶ cells / mL. 6 Cells / tubes were cryopreserved in liquid nitrogen.

[0075] 4. Stability identification of monoclonal hybridoma cell lines

[0076] The established monoclonal hybridoma cell line 1A10 was cultured continuously for 3 months and repeatedly subjected to liquid nitrogen cryopreservation and thawing to identify the stability of the hybridoma cell line. The results showed that the monoclonal hybridoma cell line had good stability and could still stably secrete monoclonal antibodies against HA1 protein.

[0077] 5. Preparation of monoclonal antibodies using in vivo ascites induction method

[0078] Multiparous female BALB / c mice were selected and injected intraperitoneally with 500 μL of sterile paraffin. One week later, the mice were injected intraperitoneally again with the monoclonal hybridoma cells 1A10 obtained above, at a dose of 2 × 10⁻⁶. 5 One cell per mouse; continue feeding for 1 week until the mouse abdomen is significantly distended and there is sufficient ascites, then aseptically extract the ascites from the mouse; after centrifugation of the extracted ascites, collect the supernatant, and purify the supernatant using A-column affinity chromatography to obtain high-purity anti-HA1 protein monoclonal antibody.

[0079] Example 3. Purification and Identification of Antibodies

[0080] 1. The ascites fluid containing the monoclonal antibody obtained in Example 2 was purified using Protein A affinity chromatography. The specific steps are as follows:

[0081] (1) Take 5 mL of the monoclonal antibody ascites obtained in Example 2, add 20 mL of PBS buffer, mix thoroughly, filter with a 0.45 μm filter, and put the sample in an ice box for later use.

[0082] (2) The Protein A affinity chromatography column was pretreated by washing with 12 column volumes of binding buffer.

[0083] (3) Slowly add the filtered peritoneal solution from step (1) into the pretreated Protein A affinity chromatography column, controlling the liquid flow rate to about 1 mL / min, so that the antibody can fully bind to the chromatography column;

[0084] (4) Continue washing the chromatography column with binding buffer until the protein indicator no longer turns blue, ensuring that unbound contaminating proteins are completely eluted.

[0085] (5) Take 2 mL centrifuge tubes and add 30 μL of 1M Tris-HCl buffer to each centrifuge tube to neutralize the eluent collected later; slowly add 1 mL of pH 3.0, 0.1M glycine solution to the chromatography column for elution, and collect the eluent at the same time; continue elution until the protein indicator no longer turns blue to ensure that the target monoclonal antibody is completely eluted;

[0086] (6) After elution, rinse the chromatography column with 10 column volumes of binding buffer. After rinsing, add 20% ethanol solution to the chromatography column and store the chromatography column upright at 4°C for later use.

[0087] (7) The collected antibody eluent was placed into a dialysis bag and dialysis was performed with PBS buffer as the dialysis solution for 48 hours. The dialysis solution was changed 2 to 3 times a day during the period to remove glycine from the eluent. After dialysis, the antibody concentration was measured, and the antibody was aliquoted and stored at -20°C. The purified monoclonal antibody was obtained by the same method as above and stored at -20°C.

[0088] The purified monoclonal antibody was analyzed by SDS-PAGE electrophoresis, and the results are as follows: Figure 3 As shown: Lane M is a pre-stained protein molecular weight standard (marker, molecular weight range 10~180 kDa), with clear step-like bands visible; comparing lane 1 (unpurified A10 sample) and lane 2 (purified 2A10 sample), the unpurified serum culture supernatant contained a large number of contaminating protein bands, while after purification by Protein G affinity chromatography, the monoclonal antibody showed typical IgG dimer characteristic bands, namely a heavy chain band with a molecular weight of approximately 55 kDa and a light chain band with a molecular weight of approximately 25 kDa, indicating that this purification method can effectively remove contaminating proteins and achieve specific enrichment of monoclonal antibodies.

[0089] 2. Monoclonal antibody titer and affinity determination

[0090] Monoclonal antibody titers were determined using an indirect ELISA method. The assay procedure was the same as that in Example 2, except for the primary antibody treatment. Specifically, the purified monoclonal antibody was serially diluted with PBS buffer starting at a ratio of 1:1000, and then added sequentially to each well of the ELISA plate (100 μL). H9N2 AIV-positive serum was used as a positive control. The plate was incubated at 37°C for 30 min. All other procedures were performed as described in Example 2. ELISA results (…) Figure 4 The results showed that the titer of monoclonal antibody A10 could reach 1:2.04×10. 6 .

[0091] Monoclonal antibody affinity was determined using an indirect ELISA method combined with regression analysis. The specific steps are as follows:

[0092] (1) Using HA1 protein at two concentrations of 2 μg / mL and 1 μg / mL as coating antigens, the indirect ELISA procedure described in Example 2 was followed for coating, incubation, and detection. The OD values ​​of each well were read. 450 Value and record;

[0093] (2) Using the OD of the first hole of each hole 450 Half the value is taken as 1 / 2 OD 450 Values, with 1 / mAb (the reciprocal of the monoclonal antibody concentration) as the x-axis and 1 / OD as the y-axis. 450 Plot a regression curve with the reciprocal of absorbance values ​​as the ordinate; and plot 1 / 2 OD... 450 Substitute the value into the regression equation to calculate the corresponding numerical value, and the reciprocal of this value is the monoclonal antibody concentration.

[0094] (3) Convert the monoclonal antibody concentration obtained above into molar concentration, substitute it into Formula 1, and calculate the affinity constant of the monoclonal antibody; Formula 1 is:

[0095] Kaff = (n-1) / 2(n[Ab']t-[Ab]t), where [Ag]t and [Ag']t represent 1 / 2 OD at the two coating concentrations, respectively. 450 The corresponding molar concentration, where n represents the ratio of antigen coating concentration; when the antibody affinity is at 10... 7 Up to 10 12 In the L / mol range, it exhibits better binding performance; detection results ( Figure 5 The results showed that the affinity of monoclonal antibody A10 reached 1.103 × 10⁻⁶. 9 L / mol.

[0096] 3. Monoclonal antibody specificity assay

[0097] The specificity of monoclonal antibody A10 was evaluated using an ELISA method, and the specific procedures were as follows: First, H9N2 AIV, H3 subtype AIV, H10 subtype AIV, and chicken infectious bursal virus were coated with CBS solution to prepare coated ELISA plates. Then, using monoclonal antibody A10 as the primary antibody and HRP-labeled goat anti-mouse IgG as the secondary antibody, incubation, washing, color development, and detection were performed according to the indirect ELISA procedure described in Example 2. The OD values ​​of different antigen-coated wells were compared. 450 The specific binding ability of monoclonal antibody A10 was assessed using the value, and the results are as follows: Figure 6 As shown.

[0098] Figure 6 The results showed that when the H9N2 subtype avian influenza virus HA1 protein was used as the antigen, the response signal (OD) of monoclonal antibody 1A10 was [not specified]. 450 The specific antigenicity of monoclonal antibody 1A10 was significantly higher than the negative background value. However, when using H3 subtype avian influenza virus HA1 protein, H10 subtype avian influenza virus HA1 protein, infectious bursal disease virus (IBDV) VP2 protein, and infectious bronchitis virus (IBV) S protein as antigens, the reaction signals of monoclonal antibody 1A10 were close to the negative background value, with no obvious specific binding signal. These results indicate that the monoclonal antibody 1A10 prepared in this invention has high antigenic specificity against H9N2 subtype avian influenza virus HA1 protein and shows no cross-reactivity with corresponding antigens of other subtypes of avian influenza viruses and other avian viruses.

[0099] Example 4: Determination of the variable region sequence of a monoclonal antibody

[0100] 1. Hybridoma cell culture and total RNA extraction

[0101] The monoclonal hybridoma cell line A10 obtained above was inoculated into 1640 medium containing 10% fetal bovine serum and cultured in a 37°C, 5% CO2 incubator according to standard procedures; the cells were cultured until the density reached approximately 3 × 10⁶ cells / year. 5 / cm 2 Subsequently, hybridoma cell line A10 was collected, and total RNA was extracted from the cells using the TRIzol reagent one-step method. The entire operation strictly followed aseptic operation procedures to avoid RNase contamination and ensure the integrity of the RNA.

[0102] 2. cDNA First-Strand Synthesis and PCR Amplification

[0103] Using extracted total RNA as a template, reverse transcriptase was used with Oligo(dT) as a primer, and the reverse transcription kit was followed to synthesize the first strand of cDNA. Using the synthesized cDNA as a template, PCR amplification was performed with specific primers. After amplification, the amplification products were subjected to 1% agarose gel electrophoresis to verify the specificity and integrity of the amplification products.

[0104] 3. Gene sequencing and sequence analysis

[0105] The VH (heavy chain variable region) and VL (light chain variable region) gene products obtained by PCR amplification were sequenced using the Sanger sequencing method. The specific operation is as follows: the purified VH and VL gene amplification products were combined with universal primers and bidirectional sequencing was performed to obtain the complete sequences of the VH and VL genes.

[0106] Sequencing results showed that the coding region sequence of the VH gene (SEQ ID NO.1) was 332 bp in length and encoded 99 amino acids (SEQ ID NO.2); the sequence of the VL gene (SEQ ID NO.3) was 318 bp in length and encoded 106 amino acids (SEQ ID NO.4).

[0107] SEQ ID NO.1 (Heavy chain variable region DNA sequence):

[0108] ATGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACATCTATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATACTAAATATGACCCGAAGTTCCAGGGCAAGGCCAC TATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCATCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTAGATATGGTAACTTCTGGTACTTCGATGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCATAA.

[0109] SEQ ID NO.2 (heavy chain variable region amino acid sequence):

[0110] MLSCTASGFNIKDIYMHWVKQRPEQGLEWIGRIDPANGNTKYDPKFQGKATITADTSSNTAYLQLISLTSEDTAVYYCARYGNFWYFDVWGQGTTVTVSS.

[0111] SEQ ID NO.3 (Light chain variable region DNA sequence):

[0112] ATGGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTA TCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATAA.

[0113] SEQ ID NO.4 (Amino acid sequence of the light chain variable region):

[0114] MVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWK .

[0115] The VH and VL gene sequences were submitted to the IMGT / V-QUEST database for comparison and analysis. The results showed that both the VH and VL genes contain complete complementarity-determining regions (CDR1, CDR2, CDR3) and framework regions (FR1, FR2, FR3, FR4) structures, as detailed in Tables 1 and 2. This confirms that the VH and VL gene sequences are functional immunoglobulin variable region sequences.

[0116] Table 1. Amino acid arrangement structure of the heavy chain variable region

[0117] FR-H1 LSCTAS SEQ ID NO.5 CDR-H1 GFNIKDIY SEQ ID NO.6 FR-H2 MHWVKQRPEQGLEWIGR SEQ ID NO.7 CDR-H2 IDPANGNT SEQ ID NO.8 FR-H3 KYDPKFQGKATITADTSSNTAYLQLISLTSEDTAVYYC SEQ ID NO.9 CDR-H3 ARYGNFWYFDV SEQ ID NO.10 FR-H4 WGQGTTVTVSS SEQ ID NO.11

[0118] Table 2. Amino acid arrangement structure of the light chain variable region

[0119] FR-L1 VLTQSPASLAVSLGQRATISYRAS SEQ ID NO.12 CDR-L1 KSVSTSGYSY SEQ ID NO.13 FR-L2 MHWNQQKPGQPPRLLIY SEQ ID NO.14 CDR-L2 LVS FR-L3 NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC SEQ ID NO.15 CDR-L3 QHIRELTR SEQ ID NO.16 FR-L4 SEGGPSWK SEQ ID NO.17

[0120] Based on obtaining the variable region sequence of the HA1 protein monoclonal antibody of the H9N2 subtype avian influenza virus, the present invention enables the preparation of the above-mentioned antibody through genetic engineering.

[0121] As can be seen from the above embodiments, the present invention provides a monoclonal antibody against the HA1 protein of H9N2 subtype avian influenza virus, its preparation method and application. This monoclonal antibody can be used for the detection and diagnosis of H9N2 subtype avian influenza virus and has good application prospects.

[0122] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against the HA1 protein of H9N2 subtype avian influenza virus, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

4.

2. A nucleic acid molecule encoding the monoclonal antibody of claim 1, characterized in that, The nucleic acid molecule contains nucleotide sequences encoding a heavy chain variable region and a light chain variable region; the sequence encoding the heavy chain variable region is shown in SEQ ID NO.1, and the sequence encoding the light chain variable region is shown in SEQ ID NO.

3.

3. The method for preparing the monoclonal antibody according to claim 1, characterized in that, Includes the following steps: S1. Immunize BALB / c mice with HA1 protein, and then fuse spleen cells of the immunized mice with SP2 / 0 myeloma cells to obtain hybridoma cells; S2. Using HA1 protein as the coating antigen, positive hybridoma cells were screened by indirect ELISA, and hybridoma cell lines that stably secrete monoclonal antibodies were obtained by subcloning. S3. The hybridoma cell line was inoculated into the peritoneal cavity of pretreated mice to induce ascites and purify it to obtain the monoclonal antibody.

4. The method according to claim 3, characterized in that, The HA1 protein is HA1 Proteins encoded by genes after codon optimization.

5. The method according to claim 3, characterized in that, The dose of HA1 protein for primary and booster immunizations was 48–52 μg independently.

6. The method according to claim 3, characterized in that, The immunization process in step S1 is as follows: HA1 protein is emulsified with Freund's complete adjuvant for primary immunization. Every 2 weeks, booster immunization is performed using the same method and dosage as the primary immunization, for a total of 3 booster immunizations. Super immunization is performed 3-5 days before cell fusion.

7. The method according to claim 4, characterized in that, The super-immune response is achieved by injecting BALB / c mice with HA1 protein without adjuvant, at a dose of 18-22 μg per mouse.

8. The method according to claim 4, characterized in that, HA1 protein and Freund's complete adjuvant were mixed and emulsified at a volume ratio of 0.8~1.2:0.8~1.

2.

9. The method according to claim 4, characterized in that, In step S3, Protein A affinity chromatography was used for purification, with PBS solution as the binding buffer, 0.1M glycine as the elution buffer, and 1M Tris-HCl as the neutralization buffer.

10. The use of the monoclonal antibody of claim 1 in the preparation of a detection kit or reagent kit for H9N2 subtype avian influenza virus.

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