Preparation method and application of NSP2 protein monoclonal antibody

Abstract

The invention discloses a preparation method and application of an NSP2 protein monoclonal antibody, NSP2 protein is obtained by inducing expression and purification of pET-32a expression plasmids, the NSP2 protein is used as an antigen to immunize BALB / c mice, and a hybridoma cell strain generating the antibody is obtained through a cell fusion technology. The anti-NSP2 monoclonal antibody hybridoma cell strain can stably secrete the NSP2 monoclonal antibody, and the NSP2 monoclonal antibody provided by the invention can specifically recognize JL580-NSP2 protein, can rapidly detect the NSP2 protein, can specifically bind to the NSP2 protein, ensures no cross reaction with CH-1a-NSP2, JXA1-NSP2 protein and the like, and is beneficial to wide application in immunological detection.

Description

Technical Field

[0001] The present invention relates to a method for preparing a monoclonal antibody and its application, and more specifically, to a method for preparing a monoclonal antibody against NSP2 protein and its application, belonging to the technical field of biological immunology. Background Art

[0002] The NSP2 protein has four main functional domains, producing seven NSP2 isoforms. The NSP2TF isoform influences PRRSV replication. NSP2 can accommodate gene insertions and deletions, making it a promising candidate for the development of recombinant marker vaccines. NSP2 participates in the assembly of the viral replication multiprotein complex and, as a cofactor for the NSP4 serine protease, participates in the assembly of polyproteins during viral replication. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for preparing and applying a monoclonal antibody against NSP2 protein, which has the technical characteristics of being able to rapidly detect NSP2 protein, having no cross-reaction with CH-1a-NSP2 and JXA1-NSP2 proteins, and being conducive to being widely used in immunological detection.

[0004] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0005] The present invention provides an NSP2 protein monoclonal antibody, wherein the NSP2 protein is the NSP2 protein of the PRRSV NADC30-like (JL580) strain, and the NSP2 protein monoclonal antibody is a JL580-NSP2 protein monoclonal antibody.

[0006] Preferably, the subtype of the monoclonal antibody against NSP2 protein is IgG1.

[0007] A method for preparing a monoclonal antibody against NSP2 protein, comprising the following steps:

[0008] Step 1) ligating the JL580-NSP2 gene fragment to the pET-32a(+) vector, using the constructed recombinant plasmid to express the exogenous protein and use it as an antigen to immunize mice, thereby obtaining immune mice and immune serum;

[0009] Step 2) preparing spleen cells from immunized mice and culturing myeloma cells, performing cell fusion, and screening using an indirect ELISA method to obtain hybridoma cell lines;

[0010] Step 3) cloning the hybridoma cell line to obtain positive monoclonal cells, and performing antibody subtype identification on the obtained NSP2 protein monoclonal antibody to obtain the NSP2 protein monoclonal antibody.

[0011] Preferably, the protein concentration of the NSP2 protein solution used as an antigen to immunize mice is 1.0 mg / mL.

[0012] Preferably, the NSP2 protein solution and Freund's adjuvant are emulsified in equal volumes and used in the step of immunizing mice, which includes performing primary immunization and booster immunization on the mice, wherein the number of booster immunizations is 3 times.

[0013] Preferably, the step of primary immunization comprises: emulsifying the NSP2 protein solution with an equal volume of Freund's complete adjuvant to obtain a first antigen stock solution, and performing primary immunization on mice using the first antigen stock solution;

[0014] The step of boosting immunization includes: emulsifying the NSP2 protein solution with an equal volume of Freund's incomplete adjuvant to obtain a second antigen stock solution, using the second antigen stock solution to immunize mice for the second and third time, and using the NSP2 protein solution directly as the third antigen stock solution to immunize mice for the fourth time.

[0015] The application of the monoclonal antibody against the NSP2 protein of the present invention is used to detect the NADC30-like-NSP2 protein.

[0016] The monoclonal antibody of the present invention is characterized in that an antibody secreted by hybridoma cell E10 is selected, and the heavy chain and light chain variable region gene sequences are sequenced to determine the monoclonal antibody gene sequence secreted by the hybridoma cell;

[0017] The amino acid sequence of the heavy chain variable region in the variable region sequence of monoclonal antibody E10 is:

[0018] QVQLQQSGAELVKPGASVKLSCKTSGYTFTNYWIQWVKQRPGQGLDWIGEIFPGIGAGYYNENF KGKATLTIDTSSSTAYMQLSSLTSEDSAVYFCARSLLGLKDYAMDYWGQGTSVTVSS;

[0019] The amino acid sequence of the light chain variable region in the variable region sequence of monoclonal antibody E10 is:

[0020] DIVMTQSHKFMSTSVGDRVSITCKASQDVGSAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTG SGSGTDFTLTIRNVQSEDLADYLCQQYSSYPFTFGSGTKLEIK.

[0021] Beneficial effects: It can specifically identify JL580-NSP2 protein, can quickly detect NSP2 protein, specifically bind to NSP2 protein, and ensure no cross-reaction with CH-1a-NSP2 and JXA1-NSP2 proteins, which is conducive to its wide application in immunological detection and other technical characteristics. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a diagram showing the results of PCR amplification of the NSP2 gene of the present invention.

[0023] Figure 2 This is a diagram for identifying the induced expression of the recombinant NSP2 protein of the present invention.

[0024] Figure 3 This is a diagram showing the purification results of the recombinant NSP2 protein of the present invention.

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

[0026] Figure 5 This is a cell picture of the present invention on the third day after fusion.

[0027] Figure 6 This is a picture taken on the sixth day after the present invention was fused.

[0028] Figure 7 This is a picture taken ten days after the present invention was fused.

[0029] Figure 8 This is a diagram of Western blotting validation of the monoclonal antibody of the present invention.

[0030] Figure 9 This is a diagram of Western blotting validation of the monoclonal antibody of the present invention.

[0031] Figure 10 This is a diagram of Western blotting validation of the monoclonal antibody of the present invention.

[0032] Figure 11 This is a diagram of Western blotting validation of the monoclonal antibody of the present invention.

[0033] Figure 12 This is a diagram of Western blotting validation of the monoclonal antibody of the present invention.

[0034] Figure 13 This is a diagram of Western blotting validation of the monoclonal antibody of the present invention.

[0035] Figure 14 This is a diagram of Western blotting validation of the monoclonal antibody of the present invention.

[0036] Figure 15 This is a diagram of Western blotting validation of the monoclonal antibody of the present invention.

[0037] Figure 16 This is a diagram of Western blotting validation of the monoclonal antibody of the present invention.

[0038] Figure 17 This is a diagram of Western blotting validation of the monoclonal antibody of the present invention.

[0039] Figure 18 This is a diagram for identifying subtypes of the monoclonal antibody of the present invention. DETAILED DESCRIPTION

[0040] The following is in conjunction with the instructions Figure 1-18 , the present invention is further illustrated, but the present invention is not limited to the following examples.

[0041] JL580-NSP2 gene fragment:

[0042]

[0043] In order to achieve rapid detection of JL580-NSP2 protein, the technical solution adopted in this application is as follows:

[0044] In a first aspect, the present application provides a monoclonal antibody against NSP2 protein, the subtype of which is IgG1.

[0045] In a second aspect, the present application provides a method for preparing a monoclonal antibody against NSP2 protein, the method comprising the following steps:

[0046] (1) The JL580-NSP2 gene fragment was connected to the pET-32a(+) vector to construct a recombinant plasmid expressing the exogenous protein (NSP2 protein solution);

[0047] (2) using the NSP2 protein solution to immunize mice to obtain immune mice and immune serum;

[0048] (3) preparing spleen cells from immunized mice and culturing myeloma cells, performing cell fusion treatment, and screening using indirect ELISA to obtain hybridoma cell lines;

[0049] (4) Cloning the hybridoma cell line to obtain positive monoclonal cells;

[0050] (5) The obtained NSP2 protein monoclonal antibody was subjected to antibody subtype identification.

[0051] The NSP2 protein monoclonal antibody subtype provided in the first aspect of the present application is IgG1. The monoclonal antibody has strong specificity and can accurately and quickly detect the JL580-NSP2 protein. It specifically binds to the NSP2 protein and ensures no cross-reaction with CH-1a-NSP2 and JXA1-NSP2 proteins, which is conducive to its wide application in immunological detection.

[0052] The second aspect of the present application provides a method for preparing a monoclonal antibody against an NSP2 protein. The method uses the NSP2 protein as a raw material to prepare the monoclonal antibody against the NSP2 protein. During the preparation process, immune spleen cells are fused with mouse myeloma cells using cell fusion technology, and these cells are screened using indirect ELISA to obtain positive monoclonal cells. The preparation method has a clear process and is easy to operate. The prepared monoclonal antibody against the NSP2 protein can specifically bind to the JL580-NSP2 protein and is guaranteed to have no cross-reaction with proteins such as CH-1a-NSP2 and JXA1-NSP2, thereby facilitating its wide application in immunological detection.

[0053] A specific embodiment: Preparation of NSP2 protein monoclonal antibody

[0054] 1. Experimental Materials

[0055] SPF female BALB / c mice aged 6 to 8 weeks were purchased from Shanghai Slake Laboratory Animal Co., Ltd.; SP2 / 0 myeloma cells were purchased from the Cell Bank of the Committee for Type Culture Collection of the Chinese Academy of Sciences; the pET-32a vector and E. coli Rosetta strain were maintained in our laboratory.

[0056] 2. Test methods

[0057] 2.1 Recombinant plasmid construction and transformation

[0058] EcoRⅠ and HindⅢ restriction sites were introduced upstream and downstream of the JL580-NSP2 gene fragment, respectively, and the fragment was ligated with the pET-32a(+) vector to construct a recombinant plasmid. An appropriate amount of the recombinant plasmid was used to transform Rosetta competent cells, and the correctly identified recombinant strain was subjected to bacterial maintenance operation.

[0059] 2.2 Inducible expression of NSP2 protein

[0060] The glycerol bacteria obtained above were inoculated into liquid LB medium containing Amp resistance at a volume ratio of 1:100 and activated at 37°C with a shaker at 220 rpm for 12 to 16 hours. The activated bacterial solution was then inoculated into liquid LB medium containing Amp for expansion culture for 3 to 4 hours until the OD value of the bacterial solution reached 0. 600 When the value reached 0.6-0.8, IPTG was added to a final concentration of 1.0 mM and the expression was induced at 16°C and 220 rpm for 12-16 h.

[0061] 2.3 Expression and identification of NSP2 protein

[0062] (1) Collecting bacteria: Collect the induced bacteria and centrifuge them at 6000 rpm for 20 min at room temperature;

[0063] (2) Washing the cells: Add sterile PBS solution to the cells and repeatedly pipette to resuspend the cells. Centrifuge at 12,000 rpm for 20 min at 4°C and discard the supernatant.

[0064] (3) Ultrasonic disruption: Add sterile PBS buffer to resuspend the cells and use an ultrasonic disruptor to perform ultrasonic disruption on ice for 20 min. Set the instrument frequency to 60%, and pause for 5 s after each 3 s ultrasonication.

[0065] (4) Separation of supernatant and precipitate: After complete ultrasonic disruption, the bacterial suspension was centrifuged at 12,000 rpm for 15 min at 4°C. The supernatant and precipitate were collected separately. The precipitate was resuspended in sterile PBS buffer and sampled for SDS-PAGE electrophoresis analysis and identification together with the bacterial suspension samples before and after induction.

[0066] 2.4 Purification of NSP2 protein

[0067] Collect the precipitate after bacterial cell disruption, resuspend it in 8M urea, and fully dissolve it at 4°C for 12 hours. Pour the completely dissolved suspension into a dialysis bag and perform gradient dialysis from high to low concentration using 8mol / L, 6mol / L, 4mol / L, 2mol / L, 1mol / L, and 0mol / L urea, changing the solution every 6 hours. When the last dialysis reaches 0mol / L urea, dialyze it overnight at 4°C. Collect the dialyzed protein solution, centrifuge it at 12000rpm for 10 minutes, collect the supernatant, take a small amount for SDS-PAGE analysis, and use the rest for nickel column purification.

[0068] (1) Sample processing: The supernatant after dialysis was collected and filtered through a 0.45 μm filter membrane to remove impurities;

[0069] (2) Equilibration: After the 20% ethanol protective solution in the column flows out, add equilibration solution with 2 column volumes, and repeat three times to completely remove the protective solution;

[0070] (3) Sample loading: Add the treated protein sample to the nickel column and incubate it on a rotary binder at 4°C for 3 h to allow the target protein to fully bind to the nickel filler. Open the lid at the bottom of the purification column and allow the liquid in the column to flow out under gravity. Collect the flow-through for subsequent analysis and detection.

[0071] (4) Washing: Add 6 column volumes of 25 mM imidazole washing solution, incubate at 4°C for 15 min, and then drain the liquid. Repeat three times to ensure that impurities are washed away. Collect the washing solution each time for subsequent analysis and detection;

[0072] (5) Elution: Add 1 to 2 column volumes of 250 mM imidazole eluent to elute the target protein, incubate at 4°C for 20 min, and allow it to slowly flow through the nickel column. Repeat three times, collecting the eluate into a different centrifuge tube each time. Collect the eluate each time for subsequent analysis and detection;

[0073] (6) Cleaning and storage of the column: Add 5 times the column volume of 500 mM imidazole washing solution and wash three times. After the washing solution is discharged, fill the nickel column with 20% ethanol preservation solution and store it at 4°C.

[0074] 2.5 Immunization of mice

[0075] Emulsify the protein prepared above with an equal volume of Freund's complete adjuvant. After sufficient emulsification, immunize 6-8 week old BALB / c mice by multiple subcutaneous injections. Immunize once every two weeks for a total of four times. Starting from the second immunization, replace the complete adjuvant with Freund's incomplete adjuvant for emulsification at a 1:1 ratio. The third immunization protocol is the same as the second. No adjuvant is added for the fourth immunization. The antigen dose per mouse is 100 μg each time.

[0076] 2.6 Immune serum titer determination

[0077] The serum antibody titer was measured by indirect ELISA 7-10 days after the third immunization. The mice with the highest antibody titer were selected for booster immunization by subcutaneous injection of 100 μg NSP2 protein.

[0078] 2.7 Preparation of Myeloma Cells

[0079] SP2 / 0 cell lines were revived using conventional cell recovery methods and cultured in a 37°C, 5% CO2 cell culture incubator. The cells were confirmed to be in a vigorous growth period and expanded 3 days before fusion. The cells were collected into centrifuge tubes on the day of fusion. The cells were washed three times with 37°C preheated RPMI-1640 medium and resuspended to prepare 2-5×10 6 cells / mouse and wait for cell fusion.

[0080] 2.8 Preparation of trophoblast cells

[0081] Peritoneal macrophages from normal mice were used as trophoblast cells. Feeder cells were prepared one day before fusion. The preparation steps of mouse peritoneal macrophages were as follows:

[0082] (1) 6-week-old healthy BALB / c mice were killed by cervical dislocation and their body surfaces were disinfected with 75% alcohol;

[0083] (2) Fix the mouse on a foam board and expose the peritoneum by blunt dissection. Use a disposable syringe to inject preheated RPMI-1640 medium into the mouse abdomen. Gently massage the mouse abdomen with the fingertips to allow the macrophages in the peritoneal cavity to fully mix into the medium. Use a syringe to extract the cell fluid in the peritoneal cavity and inject it into a centrifuge tube. Repeat the operation of extracting peritoneal macrophages three times. Centrifuge at 1000 rpm / min for 7 minutes, discard the supernatant, and add HAT selection medium containing 20% ​​fetal bovine serum to resuspend the cells.

[0084] (3) Count the cells at 1 to 2 × 10 6 Add the culture medium at a density of 100 cells / mL into a 96-well cell culture plate, add 100 μL to each well, observe the cells after 24 hours, and use them if there is no contamination.

[0085] 2.9 Preparation of splenocytes

[0086] Immunized BALB / c mice were sacrificed by eyeball blood collection, cervical dislocation, and disinfected by immersion in 75% alcohol. The mice were mounted on a sterile foam board, and the spleens were carefully removed. The spleens were crushed and ground using a disposable syringe with a removed needle to fully release splenocytes. The spleen cell suspension was then passed through a 70 μm cell strainer twice and centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded. The spleen cells were washed two to three times with prewarmed RPMI-1640 medium, resuspended, and counted using a cell counter.

[0087] 2.10 Cell Fusion

[0088] (1) Splenocytes were mixed with SP2 / 0 cells at a ratio of 10:1 to 5:1. The mixture was placed in a centrifuge tube and centrifuged at 1000 rpm for 10 min. The supernatant was discarded and the tube was washed twice with preheated RPMI-1640 medium to ensure that the splenocytes and SP2 / 0 cells were evenly mixed. All the supernatant was discarded and the bottom of the tube was gently tapped to loosen the cells.

[0089] (2) Pipette 1 mL of PEG preheated at 37°C and slowly drip the PEG while gently shaking the centrifuge tube, ensuring that the PEG is dripped in within 1 minute. Immediately place the centrifuge tube in a 37°C water bath for 1 minute.

[0090] (3) Add 1 mL of 37°C preheated RPMI-1640 culture medium within 45 seconds, add RPMI-1640 culture medium dropwise while rotating the centrifuge tube, add 2 mL of preheated RPMI-1640 culture medium within 1 minute, add 10 mL of preheated RPMI-1640 culture medium within 3 minutes, and place the centrifuge tube in a 37°C water bath for 10 minutes;

[0091] (4) Gently remove the centrifuge tube, centrifuge at 1000 rpm for 10 min, discard the supernatant, resuspend the cells in HAT selection medium, and then add 100 μL per well to the culture plate containing trophoblast cells;

[0092] (5) After 24 hours, observe for contamination. If there is no contamination, immediately return the cells to the incubator and continue culturing. After 5-7 days, half of the medium is exchanged with HAT selection medium containing 20% ​​fetal bovine serum. After 10 days after fusion, a full medium exchange is performed with HAT selection medium. After 14 days after fusion, a full medium exchange is performed with HT selection medium.

[0093] 2.11 Screening of positive hybridoma cells

[0094] When the hybridoma cell colony occupies approximately one-fifth of the well area, coat the ELISA plate with purified NSP2 protein at 5 μg / mL and incubate at 4°C overnight. 100 μL of cell culture supernatant is used as the primary antibody for ELISA assays. SP2 / 0 cell culture supernatant and normal mouse serum serve as negative controls. ELISA assay criteria are: negative wells with an OD value less than 0.1 and a P / N ratio greater than 2.1 are considered positive hybridoma wells.

[0095] 2.12 Subcloning of Positive Hybridoma Cells

[0096] Hybridoma cell subcloning is performed using the limiting dilution method, which includes the following steps:

[0097] (1) Prepare trophoblast cells one day before subcloning. Resuspend peritoneal macrophages in HT selection medium containing 20% ​​fetal bovine serum and plate 100 μL per well in a 96-well cell culture plate.

[0098] (2) Take the positive hybridoma cell suspension, add 200 μL of HT selection medium containing 20% ​​fetal bovine serum, and add it to well A1 of a 96-well plate. Then use a pipette to transfer 100 μL from well A1 to well B1 and mix gently. Repeat the 1:2 dilution throughout the column until well H1. Discard 100 μL of cell suspension and finally make the volume of all wells the same.

[0099] (3) Use an 8-channel pipette to add 100 μL of HT selection medium containing 20% ​​fetal bovine serum to all wells except the first column, then add an additional 100 μL of HT selection medium containing 20% ​​fetal bovine serum to each well in the first column, mix gently, and then use a pipette to draw 100 μL of the suspension from the first column (A1 to H1), quickly transfer it to the second column (A2 to H2), and mix gently;

[0100] (4) Repeat the 2-fold dilution in the same way throughout the 96-well plate, aspirate and discard 100 μL of the suspension from the last column (A12 to H12) to make the final volume in all wells 200 μL / well;

[0101] (5) Place the subcloned cell plate in a cell culture incubator for culture. After the cell clones are formed, the supernatant in the cell culture wells is tested by indirect ELISA. The cell culture wells that test positive need to be subcloned 2 to 3 times until the final subclone ELISA result shows a positive rate of 100%, which proves that the hybridoma cell line that can stably secrete monoclonal antibodies has been successfully established.

[0102] 2.13 Cryopreservation of Positive Hybridoma Cells

[0103] According to the steps, a cell line stably secreting anti-NSP2 monoclonal antibodies was finally obtained, and cells with a positive rate of 100% after cloning were expanded and cultured and then frozen in liquid nitrogen.

[0104] 2.14 Identification of NSP2 protein monoclonal antibody subtypes

[0105] According to the instructions of the ELISA kit for monoclonal antibody Ig class / subclass identification, the subtype of the monoclonal antibody was detected.

[0106] 3. Test results and analysis

[0107] Combined with attachment Figure 1-18 It can be seen that the monoclonal antibody secreted by the hybridoma cell line provided by the present invention has good specificity, can accurately and quickly detect the JL580-NSP2 protein, specifically binds to the NSP2 protein, and ensures no cross-reaction with CH-1a-NSP2 and JXA1-NSP2 proteins, etc., which is conducive to its wide application in immunological detection.

[0108] 3. Monoclonal Antibody Variable Region Sequence Determination

[0109] In order to determine the gene sequence of the monoclonal antibody secreted by the hybridoma cells, the antibody secreted by the hybridoma cell E10 was selected, and the gene sequences of the heavy chain and light chain variable regions were sequenced.

[0110] 3.1 Hybridoma Cell RNA Extraction

[0111] (1) Collect hybridoma cells: Wash the cells twice with PBS, remove the culture medium, and centrifuge at 2000 rpm for 5 min to retain the cell pellet.

[0112] (2) Trizol cell lysis: Add 400 μL PBS to the pellet and wash it once. After centrifugation, discard the supernatant and add 250 μL PBS to resuspend it. Then add 750 μL Trizol (flushing thoroughly) to fully lyse the cells and let it stand for 10 minutes.

[0113] (3) Chloroform extraction: Add 100 μL of chloroform substitute, shake up and down, let it stand at room temperature for 2 minutes, and then centrifuge (12000 rpm, 4℃, 15 minutes). After centrifugation, handle it with care and be careful not to mix the stratified samples again.

[0114] (4) Isopropanol precipitation: Take 400 μL of the upper aqueous phase (which is divided into three layers after centrifugation, the upper aqueous phase contains RNA, and the middle and lower layers are organic phase precipitates such as DNA and protein) and place it in a new centrifuge tube. Add 200 μL of isopropanol, invert about 10 times to mix (be careful not to shake violently), let it stand for 10 minutes, and then centrifuge (12000 rpm, 4°C, 15 minutes).

[0115] (5) Wash the precipitate with 75% ethanol: After centrifugation, discard the supernatant and add 500 μL of 75% ethanol (enzyme-free water: anhydrous ethanol = 1:3) and mix by inverting. Shake the precipitate until it is suspended and then centrifuge (12000 rpm, 4°C, 15 min).

[0116] (6) Dry and dissolve RNA: After centrifugation, discard the supernatant, centrifuge at 12000 rpm, 4°C, and leave in the air for 15 min. Use a pipette to wash away excess supernatant (do not suck up the precipitate), evaporate the ethanol, add 40 μL of DEPC water, and pipette thoroughly to dissolve.

[0117] (7) Detect RNA concentration: Use Nano nucleic acid analyzer to measure the concentration. First, adjust the volume with water, then draw 1 μL of RNA for concentration measurement.

[0118] (8) Perform reverse transcription immediately and store the remaining RNA at -80°C.

[0119] 3.2 Reverse transcription and PCR amplification

[0120] The extracted RNA was reverse transcribed using the TaKaRa reverse transcription kit using the 5'RACE technique to generate cDNA, and the nucleic acid sequences of the monoclonal antibody light and heavy chain variable regions were amplified using the upstream and downstream primers of the antibody light chain constant region and the upstream and downstream primers of the antibody heavy chain constant region, respectively.

[0121] 3.3 Gene cloning, transformation and identification

[0122] The amplified monoclonal antibody light and heavy chain variable region nucleic acid sequences were cloned into the vector pBluescript II SK(+), transformed into Escherichia coli DH5α, and single colonies were picked for PCR identification. Finally, Escherichia coli pBluescriptII SK(+)-E10-DH5α was successfully constructed.

[0123] 3.4 Plasmid extraction

[0124] (1) Preparation: Add RNase A to the Buffer P1 suspension; add anhydrous ethanol to the wash solution; and do not add precipitate to the Buffer P2 lysis solution and the P3 binding solution.

[0125] (2) Take a tube of overnight cultured bacterial solution and centrifuge it at 4°C and 8000 rpm for 2 min. Discard the supernatant.

[0126] (3) Add 250 μL of suspension to the precipitate at 4°C and shake to mix.

[0127] (4) Add 250 μL of lysis buffer, quickly and gently invert 10 times to mix, and let it stand at room temperature for 4 minutes.

[0128] (5) Add 350 μL of binding solution and mix thoroughly by inverting rapidly and gently 10 times.

[0129] (6) Centrifuge at 25°C and 12,000 rpm for 10 min. Transfer the supernatant to an adsorption column and centrifuge at 25°C and 8,000 rpm for 30 s. Discard the liquid in the collection tube.

[0130] (7) Add 500 μL of Buffer DW1, centrifuge at 25°C, 9000 rpm for 30 s, and discard the liquid in the collection tube.

[0131] (8) Add 500 μL of wash solution, centrifuge at 25°C and 9000 rpm for 30 s, discard the liquid in the collection tube, and repeat the operation once.

[0132] (9) After the alcohol evaporates, place the empty adsorption column in a centrifuge and centrifuge at 25°C and 12,000 rpm for 2 minutes.

[0133] (10) Place the empty column in a clean 1.5 mL centrifuge tube, add 50 μL of Lution Buffer to the center of the adsorption column filter membrane, let it stand at room temperature for 3 min, and then centrifuge it at 25 °C and 12,000 rpm for 1 min before collecting.

[0134] (11) Determine the plasmid concentration and send for sequencing.

[0135] result:

[0136] Monoclonal antibody E10 variable region sequence

[0137] Heavy chain variable region amino acid sequence:

[0138] QVQLQQSGAELVKPGASVKLSCKTSGYTFTNYWIQWVKQRPGQGLDWI GEIFPGIGAGYYNENFKGKATLTIDTSSSTAYMQLSSLTSEDSAVYFCARSLLG LKDYAMDYWGQGTSVTVSS.

[0139] Light chain variable region amino acid sequence:

[0140] DIVMTQSHKFMSTSVGDRVSITCKASQDVGSAVAWYQQKPGQSPKLLIY WASTRHTGVPDRFTGSGSGTDFTLTIRNVQSEDLADYLCQQYSSYPFTFGSGT KLEIK.

[0141] Finally, it should be noted that the present invention is not limited to the above embodiments and may be subject to many variations. All variations that can be directly derived or imagined by a person skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against NSP2 protein, characterized in that: The NSP2 protein is the NSP2 protein of the PRRSV NADC30-like (JL580) strain, and the NSP2 protein monoclonal antibody is the JL580-NSP2 protein monoclonal antibody.

2. A monoclonal antibody against NSP2 protein according to claim 1, characterized in that: The subtype of the monoclonal antibody against NSP2 protein is IgG1.

3. A method for preparing a monoclonal antibody against NSP2 protein according to any one of claims 1 to 2, characterized in that The preparation method comprises the following steps: Step 1) ligating the JL580-NSP2 gene fragment to the pET-32a(+) vector, using the constructed recombinant plasmid to express the exogenous protein and use it as an antigen to immunize mice, thereby obtaining immune mice and immune serum; Step 2) preparing spleen cells from immunized mice and culturing myeloma cells, performing cell fusion, and screening using an indirect ELISA method to obtain hybridoma cell lines; Step 3) cloning the hybridoma cell line to obtain positive monoclonal cells, and performing antibody subtype identification on the obtained NSP2 protein monoclonal antibody to obtain the NSP2 protein monoclonal antibody.

4. The method for preparing a monoclonal antibody against NSP2 protein according to claim 3, characterized in that: The protein concentration of the NSP2 protein solution used as an antigen for immunizing mice was 1.0 mg / mL.

5. The method for preparing a monoclonal antibody against NSP2 protein according to claim 4, characterized in that: The NSP2 protein solution and Freund's adjuvant are emulsified in equal volumes and used in the step of immunizing mice, which includes performing primary immunization and booster immunization on the mice, wherein the number of booster immunizations is 3 times.

6. The method for preparing a monoclonal antibody against NSP2 protein according to claim 5, characterized in that: The primary immunization step comprises: emulsifying the NSP2 protein solution with an equal volume of Freund's complete adjuvant to obtain a first antigen stock solution, and performing primary immunization on mice using the first antigen stock solution; The step of boosting immunization includes: emulsifying the NSP2 protein solution with an equal volume of Freund's incomplete adjuvant to obtain a second antigen stock solution, using the second antigen stock solution to immunize mice for the second and third time, and using the NSP2 protein solution directly as the third antigen stock solution to immunize mice for the fourth time.

7. Use of the monoclonal antibody against NSP2 protein according to any one of claims 1 to 6, characterized in that: Used to detect NADC30-like-NSP2 protein.

8. The monoclonal antibody according to any one of claims 1 to 6, characterized in that Select the antibody secreted by hybridoma cell E10 and sequence its heavy chain and light chain variable region gene sequences to determine the monoclonal antibody gene sequence secreted by the hybridoma cell; The amino acid sequence of the heavy chain variable region in the variable region sequence of monoclonal antibody E10 is: QVQLQQSGAELVKPGASVKLSCKTSGYTFTNYWIQWVKQRPGQGLDWIGEIFPGIGAGYYNENF KGKATLTIDTSSSTAYMQLSSLTSEDSAVYFCARSLLGLKDYAMDYWGQGTSVTVSS; The amino acid sequence of the light chain variable region in the variable region sequence of monoclonal antibody E10 is: DIVMTQSHKFMSTSVGDRVSITCKASQDVGSAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTG SGSGTDFTLTIRNVQSEDLADYLCQQYSSYPFTFGSGTKLEIK.

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