Monoclonal antibody of avian influenza virus PB2 protein, hybridoma cell, labeled compound, application and medicine

Abstract

The invention belongs to the field of biology, and discloses a monoclonal antibody of avian influenza virus PB2 protein, the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, and can specifically bind to influenza virus matrix protein PB2, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO. 2; the hybridoma cell has relatively high sensitivity and specificity, and can be specifically combined with at least five subtype avian influenza PB2 proteins; meanwhile, the invention also provides application and drugs of the labeled compound, the hybridoma cell and the monoclonal antibody.

Description

Technical Field

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

[0002] Antibody technology is mainly divided into monoclonal antibody technology and polyclonal antibody technology. Antigens are determined by multiple antigenic determinants. When an antigenic determinant stimulates the body, the antibody produced by a B lymphocyte receiving the antigen is called a monoclonal antibody. When the body is stimulated by multiple antigenic determinants, a variety of monoclonal antibodies are produced accordingly. These monoclonal antibodies mixed together are polyclonal antibodies. Compared with monoclonal antibodies, polyclonal antibodies may have multiple subtypes, resulting in poor specificity. When polyclonal antibodies are used for immunoassays, background values ​​will be generated, which will affect the experimental results to varying degrees. The high specificity and sensitivity of monoclonal antibodies make them widely used in molecular experiments and clinical diagnosis.

[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 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, D701N, etc.

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

[0005] The scheme 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 the monoclonal antibody against the PB2 protein of the 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, which can specifically bind to at least 5 subtypes of avian influenza PB2 proteins, and a hybridoma cell producing the monoclonal antibody.

[0008] At the same time, the present invention also provides the application 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 avian influenza virus PB2 protein, comprising a heavy chain variable region and a light chain variable region, capable of specifically binding to influenza virus matrix protein PB2, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1; and 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] Meanwhile, the present invention also discloses a hybridoma cell producing the monoclonal antibody as described 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 as described above in preparing a kit for detecting influenza virus, influenza virus matrix protein PB2 or influenza virus antibody, or in preparing a drug for preventing or treating influenza virus or in the quality control of influenza virus vaccine.

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

[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 an epitope located in the 627 domain, which has never been identified before. The 627 domain is a very important interval segment for cross-species transmission of avian influenza virus to infect humans. The present invention has a very important significance for identifying avian influenza viruses with cross-species infection risks by developing corresponding monoclonal antibodies.

[0019] The monoclonal antibody to the PB2 protein of avian influenza virus provided by the present invention can specifically recognize and bind to the PB2 proteins of five subtypes of avian influenza virus, has a broad spectrum among different subtypes of avian influenza virus, has a high specificity between avian influenza virus and other viruses, and has a high sensitivity. Therefore, it can be well applied to Western Blot experiments (the maximum dilution multiple 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; at the same time, 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 specific analysis results of the PB2 7B2 monoclonal antibody used for Western blot detection in Example 2 of the present invention, wherein lane 1: Mock; lanes 2-6: cell samples infected with H1N1, H3N8, H5N1, H7N9 and H9N2 subtype influenza viruses, respectively.

[0021] Figure 2 The results of the analysis 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, wherein A, B, C, D, and E are the detection results of infection with H1N1, H3N8, H5N1, H7N9, and H9N2 subtype influenza viruses, respectively, using the PB2 7B2 monoclonal antibody as the primary antibody, and 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, 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 of the analysis of the specificity of the PB2 7B2 monoclonal antibody for IP experimental detection in Example 4 of the present invention; wherein H1, H3, H5, H7 and H9 respectively represent the PB2 proteins enriched by the PB2 7B2 monoclonal antibody from samples infected with different viruses, 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 The results are analysis results of the sensitivity of the PB2 7B2 monoclonal antibody for IP experimental 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 H9N2 subtype influenza virus, PB2 is the influenza virus PB2 protein, and Actin is the internal reference protein Actin. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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: Obtaining the hybridoma cell line 7B2 and the monoclonal antibody produced by it against the PB2 protein of avian influenza virus

[0030] 1. Preparation of immunogen

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

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

[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), and primers were designed for the restriction sites BamHI and EcoRI on the vector pGEX-6P-1 with a GST tag, and the restriction sites HindIII and NdeI on the vector pET-21b with a His tag, so that the target fragments PB2-1 and PB2-2 respectively have homologous arms of the vector restriction site, and the recombinant plasmids GST-PB2-1, GST-PB2-2, His-PB2-1 and His-PB2-2 are constructed by homologous recombination.

[0034] Using the sequence of the PB2 gene of an influenza virus of the H9 subtype as a template, the PB2 protein encoding gene was amplified by PCR using the primers shown in Table 1 below and ligated to 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 transferred 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] The above-prepared immunogen was used to immunize 6-week-old BALB / c mice. The specific immunization method was as follows: for the first immunization, the antigen and the water-soluble adjuvant were mixed in a volume ratio of 9:1. After mixing evenly, the mice were subcutaneously injected at multiple points on the back of the neck, 100 μg / mouse; three weeks after the first immunization, the mice were immunized for the second time, and the method was the same as the first immunization; two weeks after the second immunization, the mice were immunized for the third time, and the method was the same as the first immunization; one week after the third immunization, the mice were blood drawn from the eye vein, the serum was separated, and the serum titer of the mice was detected by indirect ELISA method using His-tagged PB2 protein coated ELISA plate. After the titer reached the fusion requirement, the mice were boosted immunized three days before fusion (the above-prepared PB2 truncated protein immunogen was directly used to intraperitoneally inject the mice, 50 μg / mouse). After three immunizations, mice with serum antibody titers that reached the standard were selected for booster immunization.

[0044] 3. Obtaining hybridoma cells

[0045] The spleen cells of the mice after the booster immunization were fused with sp2 / 0 cells. The recombinant PB2 protein carrying the His tag expressed in the prokaryotic system was used as an antigen to establish an indirect ELISA method for screening hybridoma cells, and the screening was performed 10-14 days after the cell fusion. After three rounds of screening and subcloning of positive hybridoma cells, a hybridoma cell line that can stably secrete PB2 protein monoclonal antibodies was obtained, which was named 7B2.

[0046] 4. Detection of the titer 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 prepared at a ratio of 1:1×10 2 Start with a two-fold dilution 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 of 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. According to the protein concentration of the purified ascites, the initial concentration of the ascites was diluted to 0.01 μg / mL, and then the ascites was diluted in multiples based on the initial concentration. A total of 12 dilution gradients were performed and the reaction was carried out in an ELISA plate coated with His-PB2 protein to obtain the OD450 value at each concentration. Based on the obtained data, a scatter plot was drawn in Excel, a trend line was fitted and a curve formula was generated, and 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] In order to identify whether the antigen recognition regions of the monoclonal antibody secreted by hybridoma cell 7B2 and the monoclonal antibody of mouse ascites are in the same position, a truncated expression plasmid for PB2-2 protein (482-760aa) was constructed based on the pGEX-6P-1 expression vector. Then, multiple truncated proteins were prepared as antigens by induction through the prokaryotic expression system, and in the Western Blot detection, the mouse monoclonal antibody against GST tag (commercial) and PB2 7B2 monoclonal antibody were diluted at a ratio of 1:1000 and used as primary antibodies to detect the interaction between antigen and antibody. The results showed that the antigen recognition regions of the mouse GST monoclonal antibody and the PB2 7B2 monoclonal antibody were located in the same region, both located in the 648-664 amino acid region of the PB2 protein.

[0052] 7. Identification of subtypes of 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 cell 7B2 was sent to Beijing Qingke Biotechnology Co., Ltd. for analysis. Sequencing showed that the nucleotide sequence encoding the heavy chain variable region was shown in SEQ ID NO.3, and the nucleotide sequence encoding the light chain variable region was shown in SEQ ID NO.4; the amino acid sequence of the heavy chain variable region was shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region was shown in SEQ ID NO.2; the amino acid sequence of the heavy chain constant region was shown in SEQ ID NO.5; and the amino acid sequence of the light chain constant region was shown in SEQ ID NO.6. The heavy chain subtype was IgG1, and the light chain subtype was 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 the virus dilution solution for A549 cells in advance (add 1 mL BSA to 29 mL 1% double-antibody DMEM; if the virus is low-pathogenic IAV, add TPCK-trypsin with a final concentration of 0.6 μg / mL; if the virus is highly pathogenic IAV, TPCK-trypsin is not required);

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

[0062] c. Mix the cells in one well by blowing with 1 mL PBS, then take 20 μL of cell droplets and add them to the cell counting plate and count them using a cell counter. Combine the cell counting results and the virus infection multiplicity MOI = 1 to calculate the virus dilution multiple for a known TCID50. Based on the calculated results, use the virus diluent to dilute the virus. Note that the virus solution needs to 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 containing 5% CO2 and wait for the virus to adsorb for 1 hour;

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

[0065] (5) Sample collection: 24 hours after the virus infection, the A549 cells were collected into a 1.5 mL EP tube together with the cell supernatant. 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 boiled in a 100°C metal bath for 10 min and the cell sample was centrifuged instantaneously.

[0066] (6) Western blot identification:

[0067] Glue preparation: Add 7mL 10% SDS-PAGE separation gel to the assembled gel preparation plate, add 3mL 95% high concentration ethanol to seal the gel surface, wait for 20-30 minutes for the separation gel to solidify, pour out the ethanol solution and add SDS-PAGE concentrated gel, insert a 10-hole / 15-hole comb, and wait for the concentrated gel to solidify; then place the gel in the electrophoresis tank for assembly, fill the inner electrophoresis tank with the newly configured 1×Tris-glycine protein electrophoresis solution, and fill the outer electrophoresis tank with the old electrophoresis solution recycled before;

[0068] Sample cooking and sample loading: Take 30μL of the supernatant after ultrasonic lysis and the precipitate resuspension after PBS resuspension into a new 1.5mL EP tube, add 6μL 6×Protein loading buffer and mix well, then place in a 100℃ metal bath to cook for 10 minutes; after the sample cooking is completed, separate the sample and start preparing for sample loading. Add 10-15μL of protein sample to the loading well, and reserve at least one loading well for the protein marker;

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

[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, the PVDF membrane of the 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 (when placing each layer, a glass rod is needed to remove all bubbles). 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, you can determine whether the transfer is successful 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, take out the PVDF membrane and place it in 5% skim milk, and block it on a horizontal shaker at room temperature for 1 hour or at 4°C overnight;

[0075] (9) Incubation with primary antibody: 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) Incubation of secondary antibody: After the primary antibody incubation is completed, it is recovered and stored at 4°C. The PVDF membrane is washed 3 times with PBST solution, 5 min each time. 1 μL of HRP-labeled goat anti-mouse IgG is added to 8 mL of 5% skim milk and incubated at room temperature for 1 h on a horizontal shaker;

[0077] (11) Development and exposure: After the secondary antibody incubation is completed, wash the PVDF membrane three times with PBST solution, each time for 5 minutes. Turn on the exposure instrument at the same time as the membrane washing begins. After the membrane washing is completed, add an appropriate amount of exposure solution to the PVDF membrane until the membrane surface is completely covered. After avoiding light for 2 minutes, use tweezers to place the PVDF membrane in the exposure instrument for exposure. After the exposure is completed, 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 as described 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 antibodies used in this study were respectively diluted in 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 primary antibodies in Western Blot.

[0082] 3. Experimental results

[0083] The above Western blot specificity test results show 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 cell components, indicating that the monoclonal antibody 7B2 strain can recognize the PB2 protein of different subtypes of influenza virus through Western Blot test, and the monoclonal antibody has certain application value.

[0084] The above Western blot sensitivity test results show that the maximum dilution ratio of PB2 7B2 monoclonal antibody used in Western blot detection is 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 the virus dilution solution of MDCK cells in advance (add 1 mL BSA to 29 mL 1% double-antibody DMEM; if the virus is low-pathogenic IAV, add TPCK-trypsin with a final concentration of 2 μg / mL; if the virus is highly pathogenic IAV, TPCK-trypsin is not required);

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

[0092] c. Mix the cells in one well by blowing with 1 mL PBS, then take 20 μL of cell solution and add it to the cell counting plate and use a cell counter to count (96-well plates may not be convenient for cell counting due to their small pore size, so you can make an estimate). Combine the cell counting results and the virus infection multiplicity MOI = 0.1 to calculate the virus dilution multiple for a known TCID50. Based on the calculated results, use the virus diluent to dilute the virus. Note that the virus solution needs to be vortexed and mixed before dilution;

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

[0094] (4) Fixation: After gently shaking off the cell supernatant, add 50 μL of cell fixative to each well and let 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: Shake off the primary antibody in 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 times 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 fluorescent 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 antibodies used in this study were respectively diluted in 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, incubate the cell samples as primary antibody in IFA.

[0105] 3. Experimental results

[0106] The above IFA specific test results show 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 a certain application value in identifying various subtypes of influenza virus PB2 proteins using IFA tests.

[0107] The above IFA sensitivity test results showed that under the fluorescence microscope, the number and intensity of fluorescence in the virus-infected cell wells gradually decreased with the increase of the dilution of PB2 7B2 monoclonal antibody, and the upper limit of the dilution of 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 the virus dilution solution for A549 cells in advance (add 1 mL BSA to 29 mL 1% double-antibody DMEM; if the virus is low-pathogenic IAV, add TPCK-trypsin with a final concentration of 0.6 μg / mL; if the virus is highly pathogenic IAV, TPCK-trypsin is not required);

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

[0115] c. Mix the cells in one well by blowing with 1 mL PBS, then pipette 20 μL of cell solution into a cell counting plate and count using a cell counter. Calculate the virus dilution multiple for a known TCID50 based on the cell counting results and the virus infection multiplicity MOI = 1. Dilute the virus with virus diluent based on the calculated results. Note that the virus solution needs to 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 containing 5% CO2 and wait for the virus to adsorb for 1 hour;

[0117] (4) Fluid exchange: After virus adsorption is complete, discard the virus infection solution, gently rinse the cells once with sterile PBS, and then 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 in a 4°C centrifuge, discard the supernatant PBS, add 1 mL of ice-cold PBS again to resuspend the cells and centrifuge;

[0119] (6) Sample processing: Prepare cell lysis buffer, resuspend the cell pellet with 150 μL of cell lysis buffer, and place on ice for 30 min. Then, use an ultrasonic disruptor to disrupt the cell sample in an ice bath. After the end of ultrasound, the sample was placed in a 4℃ centrifuge at 12000rpm for 15min. After the centrifugation, the supernatant was carefully transferred to a new 1.5mL EP tube, and 20μL was taken from it as input. After adding 4μL 6×Protein loading buffer and mixing, the sample was placed in a 100℃ metal bath for 10min and stored at -20℃ for use (after taking it out from -20℃ before running the gel, it needs to be placed in a 100℃ metal bath for 5min again); the remaining 130μL was used as IP, 25μL of Protein A / G PLUS-Agarose and 10ng of 7B2PB2 monoclonal antibody (the negative control is mouse IgG) were added to it, and then RIPA lysis buffer was added to make up to a total volume of 450μL, and it was placed at 4℃ for incubation for 4-6h.

[0120] (7) Washing beads: Place the sample that has completed the incubation inversion in a 4°C centrifuge at 3000 rpm for 3 min, carefully discard the supernatant, add 400 μL of cell lysis buffer to resuspend the beads, invert at 4°C 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 precipitate 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 instantly 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 that should be used in IP experiments.

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

[0126] (2) In the step of treating the sample, 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 shows 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 an epitope located in the 627 domain, which has never been identified before. The 627 domain is a very important interval segment for cross-species transmission of avian influenza virus to infect humans. The present invention has a very important significance for identifying avian influenza viruses with cross-species infection risks by developing corresponding monoclonal antibodies.

[0132] 2. The monoclonal antibody against PB2 protein of avian influenza virus provided by the present invention can specifically recognize and bind to PB2 protein of 5 subtypes of avian influenza virus, has a broad spectrum among different subtypes of avian influenza virus, has a high specificity between avian influenza virus and other viruses, and has a high sensitivity. Therefore, it can be well applied to Western Blot experiment (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; at the same time, 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 invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A monoclonal antibody against 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. 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.

2. The monoclonal antibody according to claim 1, characterized in 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.

3. A hybridoma cell producing the monoclonal antibody according to any one of claims 1 to 3.

4. A labeling complex, characterized in that Obtained by biochemically labeling the monoclonal antibody according to any one of claims 1 to 3; The biochemical label is one or more selected from enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, and radioactive labeling.

5. Use of the monoclonal antibody according to claim 1 or 2, the hybridoma cell according to claim 3, or the labeled complex according to claim 4 in preparing a kit for detecting influenza virus, influenza virus matrix protein PB2, or influenza virus antibody.

6. Use of the monoclonal antibody according to claim 1 or 2, the hybridoma cell according to claim 3, or the labeled complex according to claim 4 in the preparation of a medicament for preventing or treating influenza virus or in the quality control of influenza virus vaccines.

7. A medicament comprising the monoclonal antibody according to claim 1 or 2.

8. A test kit for influenza virus, characterized in that: Comprising the monoclonal antibody according to claim 1 or 2 or the labeled complex according to claim 4.

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