A monoclonal antibody, hybridoma cell, labeled complex, application, and medicine for PB2 protein of avian influenza virus

Abstract

The present invention belongs to the field of biology and discloses a monoclonal antibody against the avian influenza virus PB2 protein. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, and is capable of specifically binding to the influenza virus matrix protein PB2. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2. The hybridoma cell has high sensitivity and specificity and is capable of specifically binding to at least five subtypes of avian influenza PB2 proteins. The present invention also provides applications of the labeled complex, hybridoma cells, monoclonal antibodies, and drugs.

Description

Technical Field

[0001] The present invention relates to the biological field, in particular to a monoclonal antibody, hybridoma cell, labeled complex, application and medicine of PB2 protein of avian influenza virus. Background Art

[0002] Antibody technology is primarily divided into monoclonal and polyclonal antibody technologies. An antigen is determined by multiple antigenic determinants. When a B lymphocyte responds to a single antigenic determinant and produces an antibody, it is called a monoclonal antibody. When multiple antigenic determinants are stimulated, a variety of monoclonal antibodies are produced. These monoclonal antibodies, when mixed together, form a polyclonal antibody. Compared to monoclonal antibodies, polyclonal antibodies may have multiple subtypes, resulting in lower specificity. When using polyclonal antibodies for immunoassays, background levels can be generated, which can affect experimental results to varying degrees. The high specificity and sensitivity of monoclonal antibodies make them widely used in molecular experiments and clinical diagnostics.

[0003] The PB2 protein of avian influenza virus is one of the key proteins that make up its polymerase complex. It is not only essential for the initiation of viral transcription and replication, but is also a major determinant of influenza virus virulence and mammalian adaptability. Some mutations in the PB2 protein have been identified as helping to enhance the virus's replication ability and pathogenicity in mammalian cells, such as E627K and D701N.

[0004] CN113490684A discloses monoclonal antibodies specific for the PB2 antigen of human influenza virus (FLU), nucleotide sequences, methods and kits for diagnosing infections caused by FLU.

[0005] This protocol does not specify the antigenic epitope to which it is applicable.

[0006] The technical problem to be solved by this scheme is: how to further improve the sensitivity and specificity of monoclonal antibodies against PB2 protein of avian influenza virus. Summary of the Invention

[0007] The purpose of the present invention is to provide a broad-spectrum monoclonal antibody with high sensitivity and specificity, capable of specifically binding to at least five subtypes of avian influenza PB2 proteins, and a hybridoma cell producing the monoclonal antibody.

[0008] At the same time, the present invention also provides applications of the labeled complex, hybridoma cells, nucleic acids, monoclonal antibodies, and medicines.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] A monoclonal antibody to the PB2 protein of avian influenza virus, comprising a heavy chain variable region and a light chain variable region, capable of specifically binding to the influenza virus matrix protein PB2, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2.

[0011] In the above monoclonal antibody, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.3, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.4.

[0012] At the same time, the present invention also discloses a hybridoma cell that produces the monoclonal antibody mentioned above.

[0013] In addition, the present invention also discloses a labeled complex, which is obtained by biochemically labeling the monoclonal antibody as described in any one of the above items;

[0014] Preferably, the biochemical label is one or more selected from enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, and radioactive labeling.

[0015] In addition, the present invention also discloses the use of the monoclonal antibody or hybridoma cell or labeled complex described above in preparing a kit for detecting influenza virus, influenza virus matrix protein PB2 or influenza virus antibodies, or in preparing a drug for preventing or treating influenza virus or in the quality control of influenza virus vaccines.

[0016] Finally, the present invention also discloses a drug comprising the monoclonal antibody described above and a test kit for influenza virus, wherein the test kit comprises the monoclonal antibody described above or the labeled complex described above. In addition to the monoclonal antibody against the PB2 protein of the avian influenza virus or the labeled complex against the monoclonal antibody against the PB2 protein of the avian influenza virus, the test kit for influenza virus may also contain other reagents or materials required for detection, including but not limited to a buffer and a secondary antibody.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The antigenic epitope identified by the PB2 monoclonal antibody of the present invention is located in the 627 domain, a previously unidentified region. The 627 domain is a crucial region for cross-species transmission of avian influenza viruses to humans. The development of a corresponding monoclonal antibody in this invention is of great significance for identifying avian influenza viruses with the risk of cross-species infection.

[0019] The monoclonal antibody against the PB2 protein of avian influenza virus provided by the present invention can specifically recognize and bind to the PB2 protein of five subtypes of avian influenza virus, has a broad spectrum among different subtypes of avian influenza virus, is highly specific between avian influenza virus and other viruses, and has high sensitivity. Therefore, it can be well applied to Western blot experiments (the maximum dilution factor can reach 5×10 6 times), indirect immunofluorescence assay (IFA, the maximum dilution factor can reach 5×10 5 times) and immunoprecipitation (IP, the minimum usage is only 1μg) technology, which has high application value; it also lays a technical foundation for in-depth research on the pathogenic mechanism of avian influenza virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of specificity analysis of the PB2 7B2 monoclonal antibody used in Example 2 of the present invention for Western blot detection are shown, wherein lane 1: Mock; lanes 2-6: cell samples infected with influenza viruses of H1N1, H3N8, H5N1, H7N9, and H9N2 subtypes, respectively.

[0021] Figure 2 This is the analysis result of the sensitivity of the PB2 7B2 monoclonal antibody used for Western blot detection in Example 2 of the present invention, wherein lane 1: cell sample infected with H9N2 subtype influenza virus, lane 2: Mock.

[0022] Figure 3 The results of the analysis of the specificity of the PB2 7B2 monoclonal antibody for IFA detection in Example 3 of the present invention are shown in Figures A, B, C, D, and E, which are the detection results of infection with influenza viruses of H1N1, H3N8, H5N1, H7N9, and H9N2 subtypes, respectively, using the PB2 7B2 monoclonal antibody as the primary antibody. F: Mock.

[0023] Figure 4 The results of the analysis of the sensitivity of the PB2 7B2 monoclonal antibody for IFA detection in Example 3 of the present invention are shown in Figure 3, wherein A, B, C, D, E, F, G, and H are the dilution ratios of the PB2 7B2 monoclonal antibody, which are 1:1×10 2 1:5×10 2 1:1×10 3 1:5×10 3 1:1×10 4 1:5×10 4 1:1×10 5 1:5×10 5 Detection results, I: positive control.

[0024] Figure 5 The results are the analysis results of the specificity of the PB2 7B2 monoclonal antibody used for IP experimental detection in Example 4 of the present invention; wherein H1, H3, H5, H7, and H9 represent the PB2 proteins enriched by the PB2 7B2 monoclonal antibody from samples infected with different viruses, respectively; the input is a cell sample infected with different subtypes of influenza virus; PB2 is the influenza virus PB2 protein; and Actin is the internal reference protein Actin.

[0025] Figure 6 These are the analysis results of the sensitivity of the PB2 7B2 monoclonal antibody for IP assay detection in Example 4 of the present invention; wherein IP is the PB2 protein enriched by the PB2 7B2 monoclonal antibody from samples infected with different viruses, Input is a cell sample infected with the H9N2 subtype influenza virus, PB2 is the influenza virus PB2 protein, and Actin is the internal reference protein Actin. DETAILED DESCRIPTION

[0026] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0029] Example 1: Hybridoma cell line 7B2 and its production of monoclonal antibodies against avian influenza virus PB2 protein

[0030] 1. Preparation of Immunogen

[0031] The immunogen used to immunize mice is a GST-tagged recombinant influenza virus PB2 truncated protein expressed using a prokaryotic expression system. The specific preparation method is as follows:

[0032] (1) Construction of PB2 protein prokaryotic expression vector

[0033] Using the PB2 gene sequence (SEQ ID NO. 7) of the avian influenza virus strain A / chicken / Heibei / m0530-1 / 2017 (H9N2) (abbreviated as HB17) as a template, the PB2 protein was truncated into PB2-1 and PB2-2 (PB2-1: 1-481aa; PB2-2: 482-760aa). Primers were designed for the restriction sites BamHI and EcoRI on the GST-tagged vector pGEX-6P-1, and the restriction sites HindIII and NdeI on the His-tagged vector pET-21b, so that the target fragments PB2-1 and PB2-2 respectively contained homology arms of the vector restriction sites. Recombinant plasmids GST-PB2-1, GST-PB2-2, His-PB2-1, and His-PB2-2 were constructed by homologous recombination.

[0034] Using the sequence of the PB2 gene of an H9 subtype influenza virus as a template, the PB2 protein encoding gene was amplified by PCR using the primers listed in Table 1 below, and then ligated into the pGEX-6P-1 vector.

[0035] Table 1 Primer list

[0036]

[0037]

[0038] (2) Prokaryotic expression of PB2 protein

[0039] The prokaryotic expression vector of the PB2 truncated protein constructed above was transformed into Escherichia coli DH5α competent cells, and positive strains were obtained through screening and sequencing. After the sequencing was correct, the plasmid was extracted.

[0040] (3) Preparation of PB2 protein immunogen

[0041] The successfully constructed positive plasmid was transformed into competent cells of Escherichia coli Transetta (DE3) and induced to express protein. The soluble protein expressed in the supernatant was purified and then used to immunize mice.

[0042] 2. Animal immunization

[0043] Six-week-old BALB / c mice were immunized with the immunogen prepared above. The specific immunization method was as follows: for the first immunization, the antigen and water-soluble adjuvant were mixed in a volume ratio of 9:1. After mixing evenly, the mice were injected subcutaneously at multiple points on the back of the neck at a dose of 100 μg / mouse. Three weeks after the first immunization, the mice were immunized for the second time using the same method as the first immunization. Two weeks after the second immunization, the mice were immunized for the third time using the same method as the first immunization. One week after the third immunization, blood was collected from the mice's ophthalmic vein, serum was separated, and the serum titer of the mice was tested by indirect ELISA using a His-tagged PB2 protein-coated microplate. After the titer reached the fusion requirement, the mice were boosted three days before fusion (directly using the PB2 truncated protein immunogen prepared above, injected intraperitoneally into the mice at a dose of 50 μg / mouse). After three immunizations, mice whose serum antibody titers reached the standard were selected for booster immunization.

[0044] 3. Obtaining hybridoma cells

[0045] Splenocytes from the boosted mice were fused with sp2 / 0 cells. An indirect ELISA method for screening hybridomas was established using a prokaryotically expressed His-tagged recombinant PB2 protein as an antigen. Screening was performed 10-14 days after cell fusion. After three rounds of screening and subcloning of positive hybridomas, a hybridoma cell line that stably secreted a monoclonal antibody against the PB2 protein was obtained and designated 7B2.

[0046] 4. Titer detection of monoclonal antibodies secreted by hybridoma cell 7B2

[0047] In order to determine the titer of the monoclonal antibody secreted by hybridoma cell 7B2, the indirect ELISA method established in step 3 was used to detect the antibody titer; at the same time, ascites of immunized mice was prepared as a control. The hybridoma cell supernatant was diluted with 1:1×10 2 Start with a two-fold dilution ratio of 1:5.12×10 4 times, ascites by 1:1×10 2 Start with a two-fold dilution ratio of 1:1.31×10 8 The results showed that the antibody titer of hybridoma cell supernatant can reach 1:5.12×10 4 The antibody titer in mouse ascites can reach 1:5.24×10 7 .

[0048] 5. Affinity detection of monoclonal antibodies secreted by hybridoma cell 7B2

[0049] His-PB2 protein was used as the coating antigen at a concentration of 0.1 μg / mL. Based on the protein concentration of the purified ascites, the initial concentration of the ascites was diluted to 0.01 μg / mL. Subsequently, the ascites was serially diluted based on the initial concentration, with a total of 12 dilution gradients. The reaction was carried out in an ELISA plate coated with His-PB2 protein, and the OD450 value at each concentration was obtained. Based on the obtained data, a scatter plot was drawn in Excel, a trend line was fitted, and a curve formula was generated. Half of the maximum OD450 value during the measurement process was substituted into the curve formula to calculate the antibody affinity constant Kd. The results showed that the affinity constant Kd of 7B2 was 6.940×10 -13 .

[0050] 6. Identification of the antigen recognition region of the PB2 7B2 monoclonal antibody

[0051] To determine whether the antigen-recognition regions of the monoclonal antibody secreted by hybridoma cell 7B2 and the mouse ascites monoclonal antibody are located in the same region, a truncated expression plasmid targeting the PB2-2 protein (482-760 aa) was constructed based on the pGEX-6P-1 expression vector. Subsequently, multiple truncated proteins were produced using a prokaryotic expression system. A commercially available mouse monoclonal antibody against the GST tag and the PB2-7B2 monoclonal antibody were used as primary antibodies in Western blotting to detect antigen-antibody interactions. The results showed that the antigen-recognition regions of the mouse GST monoclonal antibody and the PB2-7B2 monoclonal antibody are located in the same region, at amino acids 648-664 of the PB2 protein.

[0052] 7. Identification of subtypes of the PB2 7B2 monoclonal antibody

[0053] The subtype of the monoclonal antibody was identified using Biodragon's mouse monoclonal antibody Ig subclass identification enzyme-labeled secondary antibody ready-to-use kit (including light chain). The results showed that the subtype of the 2C5 monoclonal antibody was IgG1 with a κ light chain.

[0054] Hybridoma cells 7B2 were sent to Beijing Qingke Biotechnology Co., Ltd. for analysis. Sequencing revealed that the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.3, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.4. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.5; and the amino acid sequence of the light chain constant region is shown in SEQ ID NO.6. The heavy chain subtype is IgG1, and the light chain subtype is κ.

[0055] Example 2 Application of PB2 7B2 Monoclonal Antibody in Western Blot

[0056] 1. Specificity identification of PB2 7B2 monoclonal antibody for Western blot detection

[0057] (1) Plating: A549 cells were plated into 6-well cell plates 24 h in advance;

[0058] (2) Washing: When the cells grow to a density of about 80%-90%, discard the cell supernatant and rinse the cells three times with sterile PBS, 100 μL per well;

[0059] (3) Infection:

[0060] a. Prepare virus dilutions for A549 cells in advance (add 1 mL of BSA to 29 mL of 1% double-antibody-containing DMEM; if the virus is a low-pathogenic IAV, add TPCK-trypsin to a final concentration of 0.6 μg / mL; if the virus is a highly pathogenic IAV, TPCK-trypsin is not required).

[0061] b. Take out the required virus from the -80℃ freezer in advance and thaw it at 4℃;

[0062] c. Mix the cells from one well by pipetting with 1 mL of PBS. Pipette 20 μL of the cell solution into a cell counting plate and count using a cell counter. Calculate the virus dilution factor for a known TCID50 using the cell count results and a multiplicity of infection (MOI) of 1. Based on the calculated results, dilute the virus using virus diluent. Note that the virus solution should be vortexed and mixed before dilution.

[0063] d. Add the diluted virus to the washed 6-well plate, 1 mL per well. Place the 6-well plate in a 37°C cell culture incubator with 5% CO2 and allow the virus to adsorb for 1 hour.

[0064] (4) Medium exchange: After virus adsorption is complete, discard the virus infection solution, gently rinse the cells once with sterile PBS, and add 1 mL of virus dilution solution to each well;

[0065] (5) Sample collection: 24 hours after virus infection, A549 cells were collected together with the cell supernatant into a 1.5 mL EP tube. The tube was centrifuged at 3000 rpm for 5 min at 4°C, and the supernatant was discarded. 200 μL of protein lysis and loading buffer was added to each tube and mixed. The sample was placed in a metal bath at 100°C for 10 min, and then the cell sample was centrifuged instantaneously.

[0066] (6) Western blot identification:

[0067] Glue preparation: Add 7 mL of 10% SDS-PAGE separation gel to the assembled gel preparation plate, and add 3 mL of 95% high-concentration ethanol to seal the gel surface. After the separation gel solidifies for 20-30 minutes, pour out the ethanol solution and add SDS-PAGE stacking gel. Insert a 10-well / 15-well comb and wait for the stacking gel to solidify. Then, place the gel in an electrophoresis tank for assembly. Fill the inner electrophoresis tank with newly prepared 1× Tris-Glycine protein electrophoresis buffer and fill the outer electrophoresis tank with the previously recycled old electrophoresis buffer.

[0068] Sample cooking and loading: Transfer 30 μL of the supernatant after ultrasonic lysis and the pellet resuspended in PBS to a new 1.5 mL EP tube. Add 6 μL of 6× protein loading buffer and mix thoroughly. Place the tube in a 100°C metal bath and cook for 10 minutes. After cooking, centrifuge the sample and prepare for sample loading. Add 10-15 μL of protein sample to the loading well, leaving at least one well for the protein marker.

[0069] Electrophoresis: After sample loading is completed, set the electrophoresis instrument program as shown in Table 2 below:

[0070] Table 2 Electrophoresis program

[0071]

[0072] After 1.5 hours of electrophoresis, pay attention to the bromophenol blue line in the gel. When it approaches the bottom of the gel, turn off the instrument to complete the electrophoresis. Remove the protein gel from the gel plate and cut off the excess part of the protein gel as needed;

[0073] (7) Transfer: After the electrophoresis is completed, the protein gel is removed from the gel plate, and the excess part of the protein gel is cut off according to the experimental needs. Then, the length and width of the protein gel are measured. According to the measurement results, a PVDF membrane of appropriate size is cut and immersed in anhydrous methanol for activation; the transfer solution is pre-cooled in advance, and 4 layers of filter paper and sponge are soaked in the pre-cooled transfer solution. The transfer clip is placed with the black bottom facing down, and the sponge, filter paper, protein gel, PVDF membrane, filter paper, and sponge are laid out in order (a glass rod is needed to remove all bubbles when each layer is placed). Finally, the transfer clip is placed in the transfer tank, and an appropriate amount of transfer solution is poured in. The transfer is performed at a constant current of 250mA for 1 hour.

[0074] (8) Blocking: After the transfer is completed, the success of the transfer can be determined by observing whether there is a clear protein marker band on the PVDF membrane. If there is a clear protein marker on the PVDF membrane, remove the PVDF membrane and place it in 5% skim milk. Block it on a horizontal shaker at room temperature for 1 hour or at 4°C overnight.

[0075] (9) Incubation with primary antibodies: After blocking, discard the 5% skim milk and wash the residue on the PVDF membrane with PBST solution. Then, place the membrane in commercial rabbit PB2 monoclonal antibody and mouse GST monoclonal antibody diluted in primary antibody diluent and incubate overnight at 4°C on a horizontal shaker.

[0076] (10) Secondary antibody incubation: After the primary antibody incubation is completed, the membrane was recovered and stored at 4°C. The PVDF membrane was washed three times with PBST solution, each time for 5 min. 1 μL of HRP-labeled goat anti-mouse IgG was added to 8 mL of 5% skim milk and incubated on a horizontal shaker at room temperature for 1 h.

[0077] (11) Development and exposure: After the secondary antibody incubation is complete, wash the PVDF membrane three times with PBST solution, each time for 5 minutes. Start the exposure instrument at the same time as the membrane washing begins. After the membrane washing is complete, add an appropriate amount of exposure solution to the PVDF membrane until the membrane surface is completely covered. After protecting from light for 2 minutes, use tweezers to place the PVDF membrane in the exposure instrument for exposure. After the exposure is complete, save the image and analyze the results.

[0078] 2. Sensitivity determination of PB2 7B2 monoclonal antibody for Western blot detection

[0079] Virus infection and Western blot analysis were performed according to the method in 1 above, but the following two conditions were adjusted.

[0080] (1) The infected viruses were all A / chicken / Heibei / m0530-1 / 2017(H9N2), MOI=1;

[0081] (2) The 7B2 PB2 monoclonal antibody used in this study was diluted in a 1:1×10 3 1:5×10 3 1:1×10 4 1:5×10 4 1:1×10 5 1:5×10 5 1:1×10 6 1:5×10 6 1:1×10 7 After dilution, the cell samples were incubated as the primary antibody in Western Blot.

[0082] 3. Experimental results

[0083] The above Western blot specificity test results showed that the PB2 7B2 monoclonal antibody can specifically bind to the PB2 proteins of five influenza virus subtypes: H1N1, H3N8, H5N1, H7N9 and H9N2 ( Figure 1 ), and did not react with other cellular components, indicating that the monoclonal antibody 7B2 strain could recognize the PB2 protein of different subtypes of influenza virus through Western Blot test, and the monoclonal antibody had certain application value.

[0084] The above Western blot sensitivity test results showed that the maximum dilution ratio of PB2 7B2 monoclonal antibody used in Western blot detection was 1:5×10 6 ( Figure 2 ).

[0085] Example 3 Application of PB2 7B2 Monoclonal Antibody in Indirect Immunofluorescence (IFA)

[0086] 1. Specificity identification of PB2 7B2 monoclonal antibody for IFA detection

[0087] (1) Infecting MDCK cells with five influenza virus subtypes (H1N1, H3N8, H5N1, H7N9, and H9N2) according to the virus infection method in Example 2;

[0088] (2) Washing: When the cells grow to a density of about 80%-90%, discard the cell supernatant and wash the cells three times with sterile PBS, adding 100 μL per well each time;

[0089] (3) Infection:

[0090] a. Prepare virus dilutions for MDCK cells in advance (add 1 mL of BSA to 29 mL of 1% double-antibody-containing DMEM; if the virus is a low-pathogenic IAV, add TPCK-trypsin to a final concentration of 2 μg / mL; if the virus is a highly pathogenic IAV, TPCK-trypsin is not required).

[0091] b. Take out the required virus from the -80℃ freezer in advance and thaw it at 4℃;

[0092] c. After mixing the cells in one well with 1 mL of PBS, pipette 20 μL of cell solution into a cell counting plate and count using a cell counter (96-well plates may not be convenient for cell counting due to their small pore size, so you can estimate the number of cells). Combine the cell count results and the virus infection multiplicity of infection (MOI) of 0.1 to calculate the virus dilution factor for a known TCID50. Based on the calculated results, dilute the virus with virus diluent. Note that the virus solution needs to be vortexed and mixed before dilution.

[0093] d. Add the diluted virus to a washed 96-well plate, adding 100 μL per well. Incubate the 96-well plate in a 37°C cell culture incubator with 5% CO2 for 24 hours without changing the medium.

[0094] (4) Fixation: After gently shaking off the cell supernatant, add 50 μL of cell fixative directly to each well and let it stand at room temperature for 15 min.

[0095] (5) Washing: After drying the fixative in the cell plate, wash the cells three times with PBS, adding 100 μL per well each time;

[0096] (6) Incubation with primary antibody: dilute the 7B2 PB2 monoclonal antibody used in this study 1:1×102 times with PBS and use it as the primary antibody. Add 30 μL to each well and incubate in a 37°C constant temperature incubator for 1 h.

[0097] (7) Washing: Remove the primary antibody from the cell plate and wash the cells three times with PBS, adding 100 μL per well each time;

[0098] (8) Incubation with secondary antibody: Dilute FITC-labeled goat anti-mouse IgG fluorescent secondary antibody 400-fold with PBS as the secondary antibody, add 30 μL to each well, wrap the cell plate with tin foil to avoid light, and place it in a 37°C constant temperature incubator for another 1 h;

[0099] (9) Washing: Shake off the secondary antibody in the cell plate and wash the cells three times with PBST, adding 100 μL per well each time;

[0100] (10) Observation results: Use a fluorescence microscope to observe the cells under ultraviolet light. If the cells are observed to emit green fluorescence, they are determined to be positive cells after positive infection, and the well is a positive cell well.

[0101] 2. Determination of IFA sensitivity of PB2 7B2 monoclonal antibody

[0102] Indirect immunofluorescence identification was performed according to the method in 1 above, but the following two conditions were adjusted.

[0103] (1) The virus used in this experiment was A / chicken / Heibei / m0530-1 / 2017(H9N2), MOI=0.1;

[0104] (2) The 7B2 PB2 monoclonal antibody used in this study was diluted in a 1:1×10 2 1:5×10 2 1:1×10 3 1:5×10 3 1:1×10 4 1:5×10 4 1:1×10 5 1:5×10 5 After dilution, the cell samples were incubated as the primary antibody in IFA.

[0105] 3. Experimental results

[0106] The above IFA specificity test results showed that under infection conditions, the PB2 7B2 monoclonal antibody can specifically recognize and bind to the PB2 protein of influenza virus of H1, H3, H5, H7, and H9 subtypes. Under a fluorescence microscope, clear and obvious green fluorescence can be observed in the wells infected with influenza virus of different subtypes ( Figure 3 ). This indicates that the PB2 7B2 monoclonal antibody has certain application value in identifying PB2 proteins of various subtypes of influenza virus using IFA test.

[0107] The above IFA sensitivity test results showed that under a fluorescence microscope, the number and intensity of fluorescence in the virus-infected cell wells gradually decreased with the increase of the dilution of the PB2 7B2 monoclonal antibody, and the upper limit of the dilution of the PB2 7B2 monoclonal antibody was 1:5×10 5 ( Figure 4 ).

[0108] Example 4 Application of PB2 7B2 Monoclonal Antibody in Immunoprecipitation (IP)

[0109] 1. Specificity identification of PB2 7B2 monoclonal antibody for IP detection

[0110] (1) Plating: A549 cells were plated into 6-well cell plates 24 h in advance;

[0111] (2) Washing: When the cells grow to a density of about 80%-90%, discard the cell supernatant and rinse the cells three times with sterile PBS, 100 μL per well;

[0112] (3) Infection:

[0113] a. Prepare virus dilutions for A549 cells in advance (add 1 mL of BSA to 29 mL of 1% double-antibody-containing DMEM; if the virus is a low-pathogenic IAV, add TPCK-trypsin to a final concentration of 0.6 μg / mL; if the virus is a highly pathogenic IAV, TPCK-trypsin is not required).

[0114] b. Take out the required virus from the -80℃ freezer in advance and thaw it at 4℃;

[0115] c. Mix the cells from one well by pipetting with 1 mL of PBS. Pipette 20 μL of the cell solution into a cell counting plate and count using a cell counter. Calculate the virus dilution factor for a known TCID50 using the cell count results and a multiplicity of infection (MOI) of 1. Based on the calculated results, dilute the virus using virus diluent. Note that the virus solution should be vortexed and mixed before dilution.

[0116] d. Add the diluted virus to the washed 6-well plate, 1 mL per well. Place the 6-well plate in a 37°C cell culture incubator with 5% CO2 and allow the virus to adsorb for 1 hour.

[0117] (4) Medium exchange: After virus adsorption is complete, discard the virus infection solution, gently rinse the cells once with sterile PBS, and add 1 mL of virus dilution solution to each well;

[0118] (5) Sample collection: Discard the culture medium, blow off the cells with 1 mL of ice-cold PBS and collect them into a 1.5 mL EP tube. Centrifuge at 1000 rpm for 5 min at 4°C. Discard the supernatant PBS, add 1 mL of ice-cold PBS again to resuspend the cells and centrifuge again.

[0119] (6) Sample processing: Prepare cell lysis buffer, resuspend the cell pellet with 150 μL of cell lysis buffer, and place it on ice for 30 min. Then, use an ultrasonic disruptor to disrupt the cell sample in an ice bath. After sonication, the sample was centrifuged at 12,000 rpm in a 4°C centrifuge for 15 min. After centrifugation, the supernatant was carefully transferred to a new 1.5 mL EP tube, and 20 μL was aspirated as input. After adding 4 μL of 6× Protein loading buffer and mixing, the sample was boiled in a 100°C metal bath for 10 min and stored at -20°C for use (after being taken out of -20°C before running the gel, the sample needed to be boiled again in a 100°C metal bath for 5 min). The remaining 130 μL was used as IP, and 25 μL of Protein A / G PLUS-Agarose and 10 ng of 7B2PB2 monoclonal antibody (the negative control was mouse IgG) were added to it. Then, RIPA lysis buffer was added to the total volume of 450 μL, and the tube was incubated at 4°C with rotation for 4-6 h.

[0120] (7) Washing beads: Place the sample after incubation at 4°C in a centrifuge at 3000 rpm for 3 min, carefully discard the supernatant, add 400 μL of cell lysis buffer to resuspend the beads, place at 4°C inverted for 3 min, and then centrifuge again. Repeat the above steps 6-8 times;

[0121] (8) Sample cooking: After discarding the 400 μL cell lysate used to wash the beads for the last time, add 40 μL of cell lysate to the pellet again, add 8 μL of 6× protein loading buffer and mix well. Place the sample in a 100°C metal bath and cook for 10 min. Centrifuge immediately and take the supernatant of the IP sample and the input for Western Blot identification.

[0122] (9) Western Blot Identification: Western Blot was performed using the method in Example 2.

[0123] 2. Sensitivity determination of PB2 7B2 monoclonal antibody for IP detection

[0124] Immunoprecipitation experiments were performed as described in 1 above, but the following two conditions were adjusted to test the minimum dose of monoclonal antibody PB2 7B2 for use in IP experiments.

[0125] (1) The infected viruses were all A / chicken / Heibei / m0530-1 / 2017(H9N2), MOI=1;

[0126] (2) In the sample processing step, 10 μg, 5 μg, 2.5 μg, and 1 μg of PB2 7B2 monoclonal antibody were added to the sample, respectively, and then incubated.

[0127] 3. Experimental results

[0128] The PB2 7B2 monoclonal antibody can specifically recognize and bind to the PB2 protein of influenza virus of H1, H3, H5, H7, and H9 subtypes, and detect the PB2 protein by Western Blot experiment ( Figure 5 ). This indicates that the monoclonal antibody 7B2 strain has certain application value in IP experiments.

[0129] When the amount of PB2 7B2 monoclonal antibody added was reduced to 1 μg, the enriched PB2 protein band was almost undetectable. This also indicates that the minimum effective dose of PB2 7B2 monoclonal antibody in IP experiments is at least 1 μg ( Figure 6 ).

[0130] Result analysis:

[0131] 1. The antigenic epitope identified by the PB2 monoclonal antibody of the present invention is located in the 627 domain, a previously unidentified region. The 627 domain is a crucial region for cross-species transmission of avian influenza viruses to humans. The development of a corresponding monoclonal antibody in this invention is of great significance for identifying avian influenza viruses with the risk of cross-species infection.

[0132] 2. The monoclonal antibody against the PB2 protein of avian influenza virus provided by the present invention can specifically recognize and bind to the PB2 protein of five subtypes of avian influenza virus, has a broad spectrum among different subtypes of avian influenza virus, is highly specific between avian influenza virus and other viruses, and has high sensitivity. Therefore, it can be well applied to Western blot experiments (the maximum dilution factor can reach 5×10 6 times), indirect immunofluorescence assay (IFA, the maximum dilution factor can reach 5×10 5 times) and immunoprecipitation (IP, the minimum usage is only 1μg) technology, which has high application value; it also lays a technical foundation for in-depth research on the pathogenic mechanism of avian influenza virus.

[0133] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

Claims

1. A monoclonal antibody against the PB2 protein of avian influenza virus, characterized in that The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, and can specifically bind to the influenza virus matrix protein PB2 of five subtypes: H1N1, H3N8, H5N1, H7N9, and H9N2. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.3, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.

4.

2. Use of the monoclonal antibody according to claim 1 in preparing a kit for detecting influenza virus and influenza virus matrix protein PB2, wherein There are five subtypes of influenza viruses: H1N1, H3N8, H5N1, H7N9 and H9N2.

3. A medicament comprising the monoclonal antibody according to claim 1.

4. A detection kit for influenza virus, characterized in that: Comprising the monoclonal antibody according to claim 1.

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