Anti-giant panda VEGFA monoclonal antibody, hybridoma cell line and use thereof

Abstract

The present invention discloses an anti-giant panda VEGFA monoclonal antibody, a hybridoma cell line and its use, and belongs to the field of biotechnology. The amino acid sequence of the heavy chain variable region of the anti-giant panda VEGFA monoclonal antibody 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. The hybridoma cell line VEGF Ab was deposited in the China Type Culture Collection Center of Wuhan University, Wuhan, China on September 4, 2024, and its deposit number is CCTCC NO: C2023276. The present invention utilizes cell fusion technology to obtain a hybridoma cell line capable of secreting anti-giant panda VEGFA monoclonal antibodies, further collects and purifies the cell line culture supernatant, and obtains a highly specific VEGFA monoclonal antibody.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an anti-giant panda VEGFA monoclonal antibody, a hybridoma cell line and uses thereof. Background Art

[0002] VEGFA (Vascular Endothelial Growth Factor A) is a gene encoding vascular endothelial growth factor A. This gene encodes a protein that is a cytokine that is very important for the regulation of angiogenesis and vascular permeability. VEGFA protein promotes the proliferation and migration of vascular endothelial cells by binding to receptors on the cell surface, thus playing a key role in the angiogenesis process. In mice, VEGFA plays a key role in angiogenesis by promoting endothelial cell proliferation, migration and lumen formation. For example, in mouse models, VEGFA overexpression can lead to abnormal vascular proliferation, such as retinal angiogenesis. In pigs, VEGFA is also involved in the angiogenesis process. For example, the pork central pulmonary hypertension model showed that increased VEGFA expression was associated with pulmonary artery smooth muscle proliferation and vascular remodeling. In rats (Rattusnorvegicus), VEGFA can promote inflammatory responses and tissue repair by increasing vascular permeability. For example, rat model studies have shown that VEGFA is involved in the regulation of vascular permeability in wound healing and tissue repair. In dogs, the role of VEGFA also involves the regulation of vascular permeability. For example, in a dog model of myocardial infarction, VEGFA promoted angiogenesis and vascular permeability during myocardial reperfusion and repair. In cattle, VEGFA not only acts on angiogenesis, but also promotes the survival and proliferation of other cell types. For example, the expression of VEGFA in bovine adipocytes is associated with adipocyte proliferation and adipose tissue growth. By studying the function of VEGFA in different species, we can gain a deeper understanding of its role in biology. VEGFA plays an important role in many species, especially in vascular biology, tissue repair, and the occurrence and development of diseases. Studies on its structure and function help to deeply understand the mechanisms of angiogenesis and related diseases, and provide an important basis for the development of related therapeutic strategies.

[0003] Currently, there is a lack of specific antibodies against VEGFA in giant pandas, and research on VEGFA in giant pandas is insufficient. Therefore, it is necessary to obtain highly specific monoclonal antibodies against VEGFA in giant pandas. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides an anti-giant panda VEGFA monoclonal antibody, a hybridoma cell line and uses thereof, which can effectively solve the problem that there is no specific anti-giant panda VEGFA specific antibody in the prior art.

[0005] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0006] The object of the present invention is to provide an anti-giant panda VEGFA monoclonal antibody, 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.

[0007] Furthermore, the subtype of the anti-giant panda VEGFA monoclonal antibody is IgG2b.

[0008] Another object of the present invention is to provide a hybridoma cell line VEGF Ab (Hybridoma cell line VEGF Ab) that secretes anti-giant panda VEGFA monoclonal antibodies. The hybridoma cell line VEGF Ab was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on September 4, 2024, and its preservation number is CCTCC NO: C2023276.

[0009] Another object of the present invention is to provide an antibody composition, which comprises the above-mentioned anti-giant panda VEGFA monoclonal antibody and a pharmaceutically acceptable carrier, buffer and excipient thereof.

[0010] Another object of the present invention is to provide a kit comprising the above-mentioned anti-giant panda VEGFA monoclonal antibody or antibody composition.

[0011] Another object of the present invention is to provide the use of the above anti-giant panda VEGFA monoclonal antibody, antibody composition or kit in basic medical research for non-diagnosis / treatment purposes.

[0012] Furthermore, basic medical research is Western Blot, immunohistochemistry or flow cytometry analysis.

[0013] Another object of the present invention is to provide the use of the above anti-giant panda VEGFA monoclonal antibody or antibody composition in the preparation of a preparation for detecting giant panda VEGFA.

[0014] Beneficial effects of the present invention:

[0015] The present invention uses cell fusion technology to obtain a hybridoma cell line that can secrete anti-giant panda VEGFA monoclonal antibodies, and further collects and purifies the cell line culture supernatant to obtain a highly specific VEGFA monoclonal antibody. Then, it is applied to Western Blot protein blotting detection, laying an important foundation for the localization and tissue expression information of VEGFA, the study of VEGFA and its receptors and target cells using highly specific VEGFA monoclonal antibodies, and the study of the role of VEGFA in giant panda embryo diapause. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The expression result of VEGFA (34-516) protein was analyzed by SDS-PAGE; Band M: SDS-PAGE Protein Marker; Band 0: Control (without IPTG); Band 1: Induced at 15℃ for 16 h; Band 2: Induced at 37℃ for 16 h;

[0017] Figure 2 The figure shows the results of SDS-PAGE analysis of the purification of VEGFA (34-516) protein supernatant in inclusion bodies; where, band M: SDS-PAGE Protein Marker; band 1: supernatant after whole bacteria lysis and centrifugation; band 2: effluent after supernatant incubated with Ni-IDA; band 3: elution fraction of 50mM Imidazole; bands 4-9: elution fraction of 500mM Imidazole;

[0018] Figure 3 This is the result of Western-blot detection of giant panda VEGFA monoclonal antibody. DETAILED DESCRIPTION

[0019] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0020] Example 1

[0021] 1. Preparation of anti-giant panda VEGFA monoclonal antibody

[0022] 1.1 Immunogen preparation

[0023] 1.1.1 Preparation of VEGFA (34-516) recombinant plasmid: According to the amino acid sequence of giant panda VEGFA in the NCBI database, accession number: XP_034503844.1, the sequence is: YSFIQGLPSSFFSSAILKSTFFP VLIYFLLPVPRLNRADRLGRLLLYFPKSLRILETSREKKEVARAPERSRGRERETGSESARGRASNDRDRGKVSDLLLGVTARARREPSPSGSRSGPAALTDRQTDTAPRPSAHLLPGRRPRVDAAASRGQEPEPAPAGGVEGVGARGVAVKLFVQLLGCSRSGGAVVRAGAAEPSGTGRSASSGREEPQSEEGEEEEEKEEERGPRWRLGARKPGSWTGEAAVCADSAPAARAPQALARASAPGGRG DSLGAEESGPPRSPSRRGSASRAGSGRASETMNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGEHKPHEVVKFMDVYQRSYCRPIETLVDILQEYPDEVEYIFKPSCVPLMRCAGCCNDEGLECVPTEEFNITMQIMRIKPHQGQHIGEMSFLQHSKCECRPKKDRARQEKKSIGGKGKGQKRKRKKSRYKPWSVPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR (SEQ ID NO.3), a total of 516 amino acids. In order to obtain a specific immunogen, a VEGFA (34-516) recombinant protein expression vector expressing the amino acid sequence of VEGFA protein 34-516 was constructed by prokaryotic expression. The VEGFA (34-516) gene was inserted into the expression vector pET30a by total gene synthesis through the restriction enzyme sites NdeI and HindIII, and the accuracy of the final expression vector was confirmed by enzyme digestion and sequencing.

[0024] 1.1.2 VEGFA (34-516) protein expression and identification

[0025] The constructed plasmid containing the VEGFA (34-516) gene was transformed into BL21 (DE3) competent cells, and then evenly spread on LB plates (containing 50 μg / mL kanamycin sulfate), and then inverted in a 37°C incubator overnight.

[0026] Select a single clone from the transformed plate and inoculate it into 4 mL of LB medium (containing 50 μg / mL kanamycin sulfate) and culture until OD 600 The final concentration of IPTG was 0.2 mM, and the culture medium was placed at 15°C and 37°C to induce expression.

[0027] The induced culture medium was centrifuged at 12000 rpm for 5 min, the supernatant was removed, PBS was added to resuspend the precipitate, and finally SDS-PAGE loading buffer was added to heat the sample at 100°C for 10 min, then centrifuged and the supernatant was electrophoresed. Electrophoresis was performed at 160 V until the bromophenol blue band migrated to 1 cm from the bottom of the gel. The gel was taken out and stained and destained using the protein gel rapid processing system. The results are shown in the figure. Figure 1 .

[0028] Expand the culture and grow to OD 600 =0.8, add IPTG at a final concentration of 0.2 mM, and collect the bacteria after induction at 15°C for 16 h. 1.1.3 VEGFA (34-516) protein purification: The whole bacteria were ultrasonically lysed with 50 mM Tris (pH8.0), 300 mM NaCl, 20 mM Imidazole containing 1% Triton X-100, 1 mM DTT, and 1 mM PMSF, and the Ni-IDA affinity chromatography column was equilibrated with 50 mM Tris (pH8.0), 300 mM NaCl, and 20 mM Imidazole buffer. The target protein was then eluted with equilibration buffers containing different concentrations of imidazole, and each elution fraction was collected for SDS-PAGE analysis. The analysis results are shown in Figure 2 .

[0029] Lane 5 with relatively high purity and concentration was collected by Ni-IDA affinity chromatography purification, dialyzed into 1× PBS (pH 7.4), filtered with a 0.22 μm filter after dialysis, and frozen in aliquots at -80°C.

[0030] 1.2 Antigen immunization of mice

[0031] Use a mixture of adjuvant:antigen = 1:1 for vaccination (if the amount of antigen is not enough, it can be mixed with sodium chloride and then emulsified with the adjuvant). Use Freund's complete adjuvant for the first immunization and Freund's incomplete adjuvant for the subsequent immunization. Prepare sterilized syringes, three-way tubes, and disposable syringes before immunization. First use a sterilized syringe to absorb the antigen and NaCl into the syringe (a total of 400μL, the amount of four syringes), and then use a sterilized syringe to absorb the adjuvant into the syringe (a total of 400μL, the amount of four syringes); finally, connect the sterilized syringe to the three-way tube for emulsification (emulsification takes about 10 minutes, until it reaches an oil-in-water state), and finally transfer the fused mixture into a disposable syringe for injection.

[0032] Day 1: Intraperitoneal injection, 200 μL per animal. (Antigen amount is 100 μg / animal)

[0033] Day 14: Intraperitoneal injection, 200 μL per animal. (After that, the antigen was 50 μg / animal)

[0034] Day 21: Intraperitoneal injection, 200 μL per mouse.

[0035] Day 27: Intraperitoneal injection, 200 μL per mouse.

[0036] (Blood can be collected from the tail of mice 3 to 4 days after the third immunization at 12000rpm for 8min, and serum titer can be measured. VEGFA (34-516) protein with a coating concentration of 20ng / ml is used as antigen, and serum titer is detected by ELISA method. When the titer reaches the standard, fusion can be prepared. If the titer is not high enough, immunization needs to be continued until it reaches the standard.) The titer of immune serum is shown in the following table:

[0037]

[0038] The above results indicate that mice 1, 2, and 3 meet the immune requirements and can be used for cell fusion experiments.

[0039] 1.3 Cell fusion

[0040] 1.3.1 Recovery of myeloma cells (SP2): First, take out the frozen cells from liquid nitrogen and quickly thaw them in a 37°C water bath to loosen the cells. Then, put the cells into a 15-mL centrifuge tube and take about 5 mL of PBS. Mix well and centrifuge at 1000 rpm for 5 min. Discard the supernatant and repeat the washing of SP2 cells twice. Then, culture the cells in a culture flask, mark it, and finally culture the flask in a 37°C, 5% CO2 incubator.

[0041] 1.3.2 Passaging of SP2 cells: When the cells in the culture flask have grown to about 80% of the bottom of the flask, the cells can be passaged. Use a pipette to blow the cells down, aspirate the culture medium into a 15mL centrifuge tube, and centrifuge at 1000r / min for 5 minutes; discard the supernatant, add 5mL of PBS, blow and mix, centrifuge again and discard the supernatant, and repeat the PBS washing step twice. After washing, add 2mL of 10% complete culture medium to resuspend the cells, take an appropriate amount of cells into the culture flask, and culture it in a carbon dioxide incubator.

[0042] 1.3.3 Preparation of trophoblast macrophages (can be prepared one day before fusion): The mice were killed by cervical dislocation. When breaking the cervical vertebrae, it is important to minimize the pressure on the abdominal cavity and avoid damaging the blood vessels in the abdominal cavity to prevent the feeder cells from containing a large number of blood cells. Soak the mouse in 75% alcohol for 5 minutes, then hold the mouse tail by hand and rinse it up and down in alcohol several times. Place it in a sterile plate. Use sterile scissors to cut the skin from the back abdomen, tear the skin on both sides by hand to expose the abdomen, and be careful not to damage the peritoneum. Wipe the peritoneum with an alcohol cotton ball. Use a syringe to draw 6-8mL of incomplete culture medium containing double antibodies and inject it into the abdominal cavity (double antibodies: incomplete culture medium = 1:100). Note that when injecting, use tweezers to lift the peritoneum to avoid the needle piercing the intestine and other abdominal organs. Use a cotton ball to gently massage the abdomen for one minute, aspirate the injected culture medium, and transfer it to a centrifuge tube. Centrifuge at 1000 r / min for 5 minutes and discard the supernatant. Wash it four times with PBS. Resuspend the cells in 10% complete medium.

[0043] Add the above cell suspension to a 96-well plate, add 100 μL to each well, and finally place the 96-well plate in a CO2 incubator for culture. (It is not easy to have too many macrophages, and some of the collected cells can be discarded as needed.)

[0044] 1.3.4 Preparation of immune spleen cells

[0045] (1) Remove mouse spleen

[0046] Take mice that meet the immunization requirements. First, pay attention to aseptic operation to prevent cell contamination. After killing the mice, soak them in 75% alcohol for about five minutes, put them in a sterile plate, and place them in a location that is convenient for your operation (in a clean bench) for dissection. First, use scissors to cut a small hole in the tail of the mouse, then use your hands to cut open the fur layer, gently wipe the cut part with an alcohol cotton ball, and then use tweezers to pick up the translucent film that wraps the internal organs, cut it open, expose the spleen, gently remove the spleen, and try to remove the fat tissue on it, and wash the removed spleen in PBS.

[0047] (2) Preparation of spleen cell suspension

[0048] First, wash the spleen with PBS, rinse it about 3 times, place the spleen in a flat dish, cut the spleen into pieces as much as possible with scissors, wash and filter with PBS, avoid tissue cells, collect the separated spleen cell suspension, centrifuge at 1000r / min for 5 minutes, and discard the supernatant; then wash with 5mL PBS, centrifuge at 1000r / min for 5 minutes; repeat three times, add 2mL incomplete culture medium (DMEM) to resuspend the cells after washing, dilute the cell suspension 100 times or 1000 times for cell counting, and place the remaining cells in a 37℃ water bath for later use.

[0049] (3) Preparation of SP2 cell suspension: Use a rubber-tipped pipette to suck up the cell fluid and blow against the thin film at the bottom of the culture bottle (the cells are all suspended or slightly attached to the wall). Then use a pipette to transfer the cell fluid to a 15 mL centrifuge tube and centrifuge at 1000 r / min for 5 minutes. Discard the supernatant, add 5 mL of PBS, mix well, and centrifuge at 1000 r / min for 5 minutes. Repeat the washing twice. After washing, add 2 mL of incomplete culture medium (DMEM) to resuspend the cells. Dilute the cell suspension 100 times or 1000 times for cell counting. Place the remaining cells in a 37°C water bath for later use.

[0050] 1.3.5 Cell fusion under the action of PEG

[0051] Mix SP2 and spleen cells in a ratio of 1:4 (between 1:10 and 1:4), centrifuge at 600rpm for 3min, and discard the supernatant. Gently tap the bottom of the centrifuge tube to loosen the cell pellet slightly. Slowly add 0.6mL of 50% PEG solution preheated at 37℃ within 1min, gently shaking and tapping while adding. Let it stand for 1min after adding. 10mL of incomplete culture solution preheated at 37℃ to terminate the PEG effect, add it at a uniform speed while tapping and rotating the centrifuge tube, and let it stand for 2min after adding. Centrifuge at 800rpm for 5min, discard the supernatant; then wash twice with PBS or incomplete culture solution to remove PEG.

[0052] After washing, discard the supernatant and resuspend the cells with 10mL HAT selection medium. Add the above cells to a 96-well plate with a feeder cell layer (the macrophage plate prepared previously was used. First, aspirate the liquid in the wells, then wash once with incomplete culture medium, and aspirate the liquid), add 100μL to each well; place the culture plate in a CO2 incubator for culture. After 4 hours, add complete culture medium containing HAT (19.6mL 10% complete culture medium + 0.4mL HAT) to the wells, add 100μL to each well; place the culture plate in a CO2 incubator for culture.

[0053] 1.3.6 Selective training

[0054] (1) On the fourth day of fusion culture, replace the HAT culture medium with the semi-liquid method: use a pipette to aspirate 100 μL of the supernatant in each well of the 96-well plate, discard it, and then add new 100 μL of HAT culture medium to each well (HAT culture medium is prepared as: 10% complete culture medium: HAT = 1:50). For each plate, 200 μL of 50×HAT needs to be added to 10 mL of complete culture medium.

[0055] (2) On the seventh day of fusion culture, replace the HT culture medium with the semi-liquid method: use a pipette to aspirate 100 μL of the supernatant in each well of the 96-well plate, discard it, and then add new 100 μL of HT culture medium to each well (the preparation of HT culture medium is: 10% complete culture medium: HT=1:50). For each plate, 200 μL of 50×HT needs to be added to 10 mL of complete culture medium.

[0056] 1.3.7 Positive clone screening

[0057] Obvious cloned cells can be seen in the wells after about 7 days of culture. When the cloned cells grow sufficiently (around the 12th day), the culture medium can be aspirated to detect whether they secrete antibodies.

[0058] (1) Take the previously coated ELISA plate (coated with VEGFA (34-516), coating concentration of 20 ng / mL) out of the refrigerator and let it return to room temperature. Then add 100 μL of the culture medium to be tested to each well of the plate. The two wells are used as positive and negative controls. The negative control uses 10% culture medium, and the positive control uses 10,000-fold diluted serum (serum collected from the mouse eye socket before fusion).

[0059] (2) Incubation: Seal the plate with sealing film and incubate in a 37°C incubator for 1 hour.

[0060] (3) Washing: Carefully remove the sealing film and wash 4 times, finally removing as much water as possible.

[0061] (4) Adding dual antibody: dilute the dual antibody 10,000 times, 50 μL per well.

[0062] (5) Incubation: Seal the plate with sealing film and incubate at 37°C for 30 min.

[0063] (6) Washing: Carefully remove the sealing film and wash 4 times, finally removing as much water as possible.

[0064] (7) Color development: Add 100 μL of the color developer TMB to each well, gently shake to mix, and incubate in the box for about 10 minutes.

[0065] (8) Colorimetric assay: Add 50 μL of stop solution (stop solution = 21.5 mL of concentrated sulfuric acid diluted to 200 mL) to each well, gently shake to mix, set the wavelength of the microplate reader to 450 nm, and measure the value of each well. Select the one with the highest positive result (at least 4 times that of the negative control) as the positive clone well.

[0066] 1.3.7 Screening of anti-giant panda VEGFA monoclonal antibody hybridoma cell lines by limiting dilution method

[0067] (1) Counting of cells in positive wells: The positive clone wells obtained by screening can be subjected to limiting dilution. First, transfer the cells in the wells to a 15 mL centrifuge tube (rotate while blowing to suspend the cells), and then add 10% complete culture medium to 2 mL; then count with a counting plate, and after counting, take the cell solution containing only 1000 cells for the next experiment (since only one cell is needed for one well, one 96-well plate requires about 100 cells, and 10 plates are 1000 cells).

[0068] (2) Add the cell solution to 200 mL of complete culture medium, mix well, and load the sample into a 96-well plate at 200 μL / well, for a total of ten 96-well plates.

[0069] (3) Finally, place the culture plate in a CO2 incubator for culture.

[0070] (4) After 4-5 days of culture, small cell clones can be seen under an inverted microscope. Observe the cell growth and record the wells where single cells grow and aggregate.

[0071] (5) On the fifth day of culture, change the medium in the wells where single cell growth and aggregation were recorded, and add 100 μL / well of 10% complete culture medium.

[0072] (6) On the 8th to 9th day, cell clones are visible to the naked eye, and antibody detection is carried out in time. The wells where single cells grow and aggregate and the growth conditions are relatively good are tested for culture fluid (ELSIA detection). The wells with strong positive results are anti-giant panda VEGFA monoclonal antibody hybridoma cell lines. The present invention finally screened out an anti-giant panda VEGFA monoclonal antibody hybridoma cell line VEGF Ab.

[0073] 1.3.8 Subtype identification

[0074] The isotype identification was performed using the Pierce Rapid ELISA Mouse mAb Isotyping Kit 37503. The specific procedure is as follows:

[0075] (1) Preparation: Dissolve TBS in the kit in 500 mL of double distilled water for sample dilution. Mix 870 mL of double distilled water with 30 mL of 30X Wash Buffer for plate washing. Determine the number of plates required based on the amount of samples. Store the rest in a 4°C refrigerator. Prepare 450 μL of sample diluent. Pipette 20 μL of cell culture medium into 980 μL of TBS and mix.

[0076] (2) Experimental steps: Equilibrate the plate to room temperature, add the sample to be tested to each well, 50 μL / well, and add 8 wells for each sample, i.e. one strip. Add 50 μL / well of Goat Anti-Mouse IgG+IgA+IgM HRP, gently shake the plate to mix, cover with a sealing film, incubate at room temperature for one hour, wash the plate 4 times, tap off the water, add 75 μL / well of TMB colorimetric solution for color development, and you will see that the liquid in the well turns blue. After 5-15 minutes of color development, add 75 μL / well of stop solution to terminate the reaction, and the liquid turns from blue to yellow. The antibody subtype secreted by the anti-giant panda VEGFA monoclonal antibody hybridoma cell line VEGF Ab obtained through identification and screening is IgG2b.

[0077] 1.3.9 Expansion, purification and concentration of monoclonal antibodies.

[0078] (1) Batch culture: Transfer the wells of cells that have undergone subtype identification and have produced monoclonal antibodies to a 24-well plate (rotate and pipette to suspend the cells for complete transfer) and add 600 μL of 10% complete culture medium for culture.

[0079] (2) Observe the growth of cells. When they grow to a certain size, measure their titer. Transfer cells with high titer to small culture flasks for culture (first, blow up the cells in the 24-well plate to suspend them, then use a pipette to transfer the cells to the culture flask and add 7 mL of 10% complete culture medium).

[0080] (3) Observe the growth of cells. When they grow well, transfer them to a large culture bottle for culture. Transfer one small culture bottle to two large culture bottles for culture (cell passage).

[0081] (4) You can culture several more culture bottles, freeze a portion of the cells, and use the culture fluid to purify the antibody through a column. The column used is Pierce Protein G Agarose, and the purified anti-giant panda VEGFA monoclonal antibody is concentrated using a 10000kDa ultrafiltration tube and stored at -20 degrees for later use.

[0082] 1.3.10 Cryopreservation of monoclonal antibody cell lines

[0083] After the identified anti-giant panda VEGFA monoclonal antibody hybridoma cell line VEGF Ab is cultured stably, the cells in the culture flask are blown to suspend the cells in the culture medium (the cells are generally suspended in the culture medium or grow attached to the wall), and then the cells are transferred to a 15mL centrifuge tube. Centrifuge, 1000 rpm / 5 minutes. Wash twice with PBS: first aspirate the supernatant in the centrifuge tube, discard it, add PBS, mix, centrifuge 1000 rpm / 5 minutes, and repeat once. Finally, aspirate the supernatant with a pipette, and then add an appropriate amount of freezing solution to the cells (freezing solution = 5mL serum + 4mL DMEM + 1mL DMSO, invert and mix, filter and set aside), mix. Finally, add to the freezing tube, 1mL cell solution per tube. Place it in a cryopreservation box and place it at -80°C overnight. Then place the cell line VEGF Ab in liquid nitrogen for long-term storage and use. It was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on September 4, 2024, and its preservation number is CCTCC NO: C2023276.

[0084] Example 2 Determination of titer of anti-giant panda VEGFA monoclonal antibody and its application in western blot technology

[0085] 1. Application of anti-giant panda VEGFA monoclonal antibody in western blot technology.

[0086] (1) Extraction of total protein from target cells

[0087] The total protein of giant panda liver tissue was extracted using a protein extraction kit: C510004-0020, provided by Sangon Biotech (Shanghai) Co., Ltd., and the total protein concentration was determined by the BCA method.

[0088] (2) Gel preparation: Prepare polyacrylamide gel, with 5% concentrating gel and 12% separating gel.

[0089] (3) Sample preparation: The protein loading amount is 80 μg.

[0090] (4) Electrophoresis: Concentrating gel at 80 V for 30 min; Separating gel at 120 V for 90 min.

[0091] (5) Transfer: 250 mA, 40 min.

[0092] (6) Sealing: 5% skim milk, slow shaking at 37℃ for 2 h.

[0093] (7) Incubation with primary antibody: Hybridoma cell line VEGF Ab antibody and GAPDH internal reference antibody were diluted 1:1000, incubated at 4°C with slow shaking overnight, and rewarmed at 37°C for 1 h the next day.

[0094] (8) Incubation with secondary antibody: 1:8000 dilution, incubate at 37 °C for 1 h.

[0095] (9) ECL exposure, western blot detection see Figure 3 .

[0096] Figure 3 The Western blot results showed that VEGF Ab appeared specific target bands in liver tissue and could be used in the application of western blot technology.

[0097] 2. Anti-giant panda VEGFA monoclonal antibody titer determination and sequence determination

[0098] Indirect ELISA was used to detect the titer of VEGFA monoclonal antibody purified from the culture supernatant. The specific steps were as follows:

[0099] (1) The purified VEGFA (34-516) protein in Example 1 was coated on an ELISA plate at 2 μg / mL, 50 μL / well, and incubated at 4°C overnight.

[0100] (2) Wash the coated plate, add 200 μL / well of PBS containing 2% BSA, and block at 37°C for 2 h.

[0101] (3) After washing, add LIF monoclonal antibody at dilutions (1000-fold, 2000-fold, 4000-fold, 8000-fold, 16000-fold, 32000-fold, 64000-fold, 128000-fold, 256000-fold, 512000-fold) and PBS wells as controls, and incubate at 37°C for 1 h.

[0102] (4) After washing the plate, add 1:5000 horseradish peroxidase-labeled goat anti-mouse antibody and incubate at 37°C for 1 h.

[0103] (5) Add 100 μL / well of TMB chromogenic substrate and react for 5 min.

[0104] (6) Add 50 μL of stop solution to terminate the reaction.

[0105] (7) Read the OD value of each well at 450 nm on a microplate reader.

[0106] The test results are shown in Table 1. As shown in Table 1, the titer of LIF antibody purified from the culture supernatant reached 10 5 The above results indicate that the antibody titer is high.

[0107] Table 1 Antibody titer

[0108]

[0109] 3. The obtained anti-giant panda VEGFA monoclonal antibody was sent to General Biotechnology (Anhui) Co., Ltd. for sequencing. The heavy chain variable region sequence of the monoclonal antibody is as follows:

[0110] EVQLEESGGGLVQPGGSMKLSCVASGFTFSDNWMNWVRQSPEKGLDWVAEIRLKSQNYATHYAESVKGRFIISRDDSKSTVYLQMNNLRTEDTGIYYCAGYGFAYWGQGTLVTVSA (SEQ ID NO.1)

[0111] The light chain variable region sequence is as follows:

[0112] DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYTYPYTFGSGTKLEI (SEQ ID NO.2)

[0113] In summary, the anti-giant panda VEGFA monoclonal antibody developed in the present invention can accurately identify the target protein VEGFA, laying a foundation for the localization and tissue expression information of the VEGFA protein and the study of VEGFA and its receptors and target cells using the highly specific monoclonal antibody of VEGFA, and exploring the role of VEGFA in angiogenesis in giant pandas.

[0114] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. An anti-giant panda VEGFA monoclonal antibody, characterized in that: 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.

2. The anti-giant panda VEGFA monoclonal antibody according to claim 1, characterized in that: The subtype of the anti-giant panda VEGFA monoclonal antibody is IgG2b.

3. A hybridoma cell line VEGFAb secreting the anti-giant panda VEGFA monoclonal antibody according to claim 1 or 2, characterized in that: The hybridoma cell line VEGF Ab was deposited in the China Type Culture Collection, Wuhan University, Wuhan, China on September 4, 2024, and its deposit number is CCTCC NO: C2023276.

4. An antibody composition, characterized in that The invention comprises the anti-giant panda VEGFA monoclonal antibody according to claim 1 or 2 and a pharmaceutically acceptable carrier, buffer and excipient thereof.

5. A kit, characterized in that: It comprises the anti-giant panda VEGFA monoclonal antibody according to claim 1 or 2 or the antibody composition according to claim 4.

6. Use of the anti-giant panda VEGFA monoclonal antibody according to claim 1 or 2, the antibody composition according to claim 4, or the kit according to claim 5 in basic medical research for non-diagnosis / treatment purposes.

7. The use according to claim 6, characterized in that The basic medical research is Western Blot, immunohistochemistry or flow cytometry analysis.

8. Use of the anti-giant panda VEGFA monoclonal antibody according to claim 1 or 2 or the antibody composition according to claim 4 in the preparation of a preparation for detecting giant panda VEGFA.

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