CN107056942A - Antiplatelet integrin glycoprotein I IIa monoclonal antibodies 5A10 and preparation method and application - Google Patents

Abstract

The invention discloses an anti-platelet integrin glycoprotein IIIa monoclonal antibody 5A10 and its preparation method and application. The preparation method of the anti-platelet integrin glycoprotein IIIa monoclonal antibody 5A10 is to use integrin glycoprotein IIIa peptide Fragment-coupled keyhole limpet hemocyanin KLH, then immunized BALB/c mice, and then fused the splenocytes of immunized mice with myeloma cells SP2/0. After screening, subcloned to obtain the monoclonal secretion of anti-platelet integrin glycoprotein IIIa Antibody to the hybridoma cell line HYD‑5A10. HYD‑5A10 was produced from ascites and purified on a proteinG column to obtain the desired monoclonal antibody 5A10. The monoclonal antibody 5A10 of the present invention can oxidize and crack platelets and dissolve platelet clots. The monoclonal antibody 5A10 of the present invention can also bind to human umbilical vein endothelial cells and inhibit endothelial cell proliferation, migration and angiogenesis. Therefore, the monoclonal antibody 5A10 of the present invention can be simultaneously applied to the preparation and treatment of arterial thrombosis diseases and anti-tumor angiogenesis.

Description

Anti-platelet integrin glycoprotein IIIa monoclonal antibody 5A10 and its preparation method and application

technical field

The invention relates to the fields of immunology and biomedicine, in particular a hybridoma method is used to produce a monoclonal antibody 5A10 capable of rapidly cleaving platelet clots and inhibiting vascular endothelial neogenesis, its preparation method and application.

Background technique

Arterial thrombosis is a common pathophysiological mechanism in cardiovascular, cerebrovascular, and peripheral arterial diseases. It is extremely harmful to human health and can cause ischemia and hypoxic necrosis in corresponding organs, especially the heart and brain, and endanger life. Platelets play a key role in the process of arterial thrombosis. After atherosclerotic plaque injury or rupture, platelets can be induced to adhere and gather in the damaged endothelium, forming thrombus and blocking blood vessels. Antiplatelet drugs currently commonly used in clinical practice: such as thromboxane inhibitors (aspirin), ADP receptor inhibitors (clopidogrel), platelet membrane glycoprotein IIb/IIIa (GPIIb/IIIa) receptor inhibitors (abciximab) ) and so on prevent platelet activation and clot formation by inhibiting the adhesion, activation and aggregation of normal platelets. But it has no effect on the formed platelet clot. Therefore, discovering new drug action modes will have important clinical significance and social value.

Integrins include a and β subunits. Integrin aIIbβ3 exists on platelets, while integrin avβ3 exists on vascular endothelial cells. What they share is the integrin β3 subunit. Integrin aIIbβ3 on platelets is related to platelet adhesion, activation, aggregation and thrombus formation. Integrin avβ3 on vascular endothelial cells is related to biological characteristics such as proliferation and apoptosis of endothelial cells. In previous studies, a special class of anti-platelet integrin β3 (GPIIIa) antibodies was found. After it binds to the platelet membrane GPIIIa49-66 epitope, it can activate the ROS oxidation pathway in the platelet and oxidize and break the platelet. Animal experiments have found that this type of antibody (polyclonal antibody or phage single-chain fragment form) can dissolve platelet clumps in arterial thrombus, and reduce the size of cerebral infarction in stroke rats.

Contents of the invention

The platelet membrane GPIIIa49-66 immune epitope has a high homology between human and mouse, which can be as high as 83%. When normal BALB/C mice were immunized with GPIIIa49-66 fragment, the corresponding monoclonal antibody could not be obtained. The purpose of the present invention is to prepare for the first time an anti-platelet integrin sugar capable of rapidly cleaving platelet clots and inhibiting vascular endothelial neogenesis by immunizing integrin GPIIIa-deficient BALB/c mice with GPIIIa49-66 fragments and combining hybridoma technology. Protein IIIa monoclonal antibody 5A10; 5A10 can lyse platelet clots and also inhibit the proliferation, migration and cast formation of human umbilical vein vascular endothelial cells (HUVEC). It can be used to prepare medicines for treating arterial thrombosis diseases and tumor diseases. The preparation method of the monoclonal antibody 5A10 is rapid, stable and suitable for industrial production.

The concrete technical scheme that realizes the object of the invention is:

An anti-platelet integrin glycoprotein IIIa monoclonal antibody 5A10 is characterized in that the monoclonal antibody 5A10 is prepared by immunizing integrin GPIIIa-deficient BALB/c mice with platelet integrin GPIIIa49-57 fragments and using hybridoma method Obtained, the heavy chain of the antibody is IgG1 type, the light chain is κ type, and the molecular weight of the antibody is 150KDa.

A method for preparing the anti-platelet integrin glycoprotein IIIa monoclonal antibody 5A10, the method comprising the following steps:

a) Coupling the chemically synthesized platelet integrin GPIIIa49-57 small peptide (CAPESIEFP fragment) with keyhole limpet hemocyanin (KLH), and immunizing integrin GPIIIa-deficient BALB/c mice by subcutaneous multi-point immunization; the antigen was used in PBS Dilute to 0.3mg/mL, mix 1000uL with 1000uL Freund's complete adjuvant or Freund's incomplete adjuvant, emulsify, subcutaneously immunize at multiple points; immunize once every 2 weeks, a total of 4 times; after the last immunization, the small Orbital venous blood was collected from the mice, serum was collected, and the titer of mouse serum was detected by Elisa method; qualified mice that were positive in the Elisa test were selected, and 50ug of each antigen was directly injected intraperitoneally into qualified mice, and cell fusion was performed three days later.

b) Splenocytes from immunized mice were fused with myeloma cells (SP2/0 cells), screened by HAT selective medium, and subcloned to obtain a hybridoma cell line with anti-platelet integrin GPIIIa49-57 monoclonal antibody, named: HYD -5A10; wherein, H of HAT: hypoxanthine A: aminopterin T: thymine nucleotide.

c) The hybridoma cell line HYD-5A10 was inoculated into mice that had been injected intraperitoneally with paraffin oil in advance, and the growth status of the mice was observed every day after the inoculation of HYD-5A10 cells. The ascitic fluid was aspirated and purified through a protein G column to obtain the anti-platelet integrin glycoprotein IIIa monoclonal antibody 5A10; wherein, the inoculum amount of HYD-5A10 was 1-2×10 ⁶ cells/mouse.

An application of the anti-platelet integrin glycoprotein IIIa monoclonal antibody 5A10 in the preparation of a drug for treating arterial thrombosis.

An application of the anti-platelet integrin glycoprotein IIIa monoclonal antibody 5A10 in the preparation of anti-tumor drugs.

Beneficial effects of the present invention:

1) The monoclonal antibody 5A10 obtained by the preparation method of the present invention can lyse platelet clots and is used for the treatment of arterial thrombosis diseases. In addition, such antibodies can break up platelets/activated platelets outside of tumor clots for the prevention and treatment of tumors.

2) The monoclonal antibody 5A10 obtained in the present invention can also inhibit the proliferation, migration and cast formation of human umbilical vein endothelial cells (HUVEC), and has a potential anti-tumor angiogenesis effect.

Description of drawings

Fig. 1 is the flow chart of preparation method of the present invention;

Fig. 2 is the binding figure of monoclonal antibody 5A10 of the present invention and platelet;

Fig. 3 is the binding diagram of monoclonal antibody 5A10 of the present invention and endothelial cells;

Figure 4 is a diagram of the depolymerization of platelet clots induced by the monoclonal antibody 5A10 of the present invention;

Figure 5 is a graph showing that the monoclonal antibody 5A10 of the present invention inhibits HUVEC proliferation;

Figure 6 is a graph showing the apoptosis of HUVEC induced by the monoclonal antibody 5A10 of the present invention;

Fig. 7 is a graph showing inhibition of HUVEC migration by the monoclonal antibody 5A10 of the present invention;

Fig. 8 is a graph showing inhibition of HUVEC angiogenesis by the monoclonal antibody 5A10 of the present invention.

detailed description

The present invention will be further described in detail in conjunction with the following specific embodiments and accompanying drawings. The process, conditions, reagents, experimental methods, etc. for implementing the present invention are general knowledge and common knowledge in the art except for the content specifically mentioned below, and the present invention has no special limitation content.

Example 1

Referring to Fig. 1, the preparation of a kind of anti-platelet integrin glycoprotein IIIa49-57 (GPIIIa49-57) mouse-derived monoclonal antibody 5A10 of the present invention comprises the following specific steps:

A. Immunization of mice

Select 10 healthy 6-8-week-old female BALB/C mice, and use KLH-GPIIIa49-57 (CAPESIEFP) as an antigen to immunize the mice by subcutaneous multi-point immunization, specifically:

On the first day, the antigen was diluted to 0.3mg/ml with PBS, and 1000ul of each antigen was mixed with 1000ul of complete Freund's adjuvant, emulsified, and subcutaneously injected into 10 mice;

On the fifteenth day, the antigens were diluted with PBS to 0.3mg/ml, and 1000ul of each antigen was mixed with 1000ul of Freund's incomplete adjuvant, emulsified, and subcutaneously injected into 10 mice;

On the twenty-ninth day, the antigen was diluted to 0.3mg/ml with PBS, and 1000ul of each antigen was mixed with 1000ul of Freund's incomplete adjuvant, emulsified, and subcutaneously injected into 10 mice;

On the 36th day, blood was collected from orbital veins of 10 mice, serum was collected, and the titer of mouse serum was detected by Elisa method;

On the forty-third day, the antigen was diluted to 0.3mg/ml with PBS, and 1000ul of each antigen was mixed with 1000ul of complete Freund's adjuvant, emulsified, and subcutaneously injected into 10 mice;

Qualified mice that were positive in the Elisa test were selected, and 50ug of the antigen was directly injected intraperitoneally into the qualified mice, and cell fusion was performed three days later.

B. Cell fusion and screening

HAT selective medium screening method: After cell fusion, if only considering the fusion of at most two types of cells, it can be divided into 5 cases: effector B cells and their own fusion cells, myeloma cells and their own fusion cells, and hybridomas cell. In the screening of hybridoma cells, the HAT (H: hypoxanthine A: aminopterin T: thymidine nucleotide) selective medium screening method is used, which is based on the inosine nucleotide in the DNA synthesis process. And pyrimidine nucleotide biosynthetic pathway designed, DNA synthesis has two pathways, the de novo synthesis pathway (D pathway) and the salvage synthesis pathway (S pathway). Among them, the D pathway synthesizes nucleotides from amino acids and other small molecular compounds, providing raw materials for the synthesis of DNA molecules. In this process, folic acid participates as an important coenzyme, and aminopterin in HAT medium is an antagonist of folic acid , can block the D pathway, the S pathway uses hypoxanthine guanine phosphoribosyltransferase (HGPRT) and thymidine kinase (TK) to catalyze hypoxanthine and thymidine to generate the corresponding nucleotides, two Enzymes are indispensable. Therefore, the D pathway of effector B cells and their own fusion cells is blocked by aminopterin. Although the S pathway is normal, it cannot reproduce indefinitely, and generally the cells will die after a few days of growth. Myeloma cells and their own fusion cells are HGPRT-deficient without the S pathway, and the D pathway is blocked by aminopterin. Therefore, only hybridoma cells have both the S pathway and can reproduce indefinitely.

1) Collection of myeloma cells, namely SP2/0 cells

①Select SP2/0 cells in good growth state (cells in the logarithmic growth phase should be selected, and the medium should be changed the day before fusion), blow the cells down with a gun, collect them in a 50ml centrifuge tube, and centrifuge at 1200rpm for 5min.

② Discard the supernatant, resuspend the cells with 20ml DMEM medium, count, take ³ ×107 cells, and centrifuge at 1200rpm for 5min.

③ Discard the supernatant, resuspend the cells with 20ml DMEM medium, and put them in the incubator for later use.

2) Collection of splenocytes

① Remove the mouse’s eyeball and take blood (put it at 37°C for 1 hour and then centrifuge it at 8000rpm for 10min. Sterilize in ethanol for 5 minutes.

②Transfer the sterilized mouse into the ultra-clean bench, with the left side up, cut a small opening in the skin, pull the skin from the opening to expose the peritoneum, cut the peritoneum with sterile surgical scissors, take out the spleen and put it in the screen.

③ Grind the spleen in a sieve, wash the sieve with 15ml DMEM medium to collect spleen cells, and centrifuge at 1200rpm for 5min.

④ Discard the supernatant and resuspend the cells in 10ml DMEM medium.

3) cell fusion

①Mix splenocytes and SP2/0 cells, and centrifuge at 1200rpm for 5min.

②Discard the supernatant (note that the supernatant should be poured out as clean as possible, you can gently invert the nozzle, and use an alcohol cotton ball to blot up a little liquid remaining in the nozzle), loosen the cells, and absorb 1ml of the preheated fusion solution. Add the reagent (PEG1450) drop by drop within 1 minute, and then place it in a 37°C water bath for 1 minute, then add the stop solution (add 25ml DMEM medium within 5 minutes, and gently stir the cells), and centrifuge at 1200rpm for 5min.

③ Discard the supernatant, add 100ml of preheated HAT medium, resuspend the cells, and mix gently (use a pipette to suck up the cells from the bottom, gently swirl and drop them in from the top of the liquid). Add the feeder cells into the 96-well plate pre-inoculated with a row gun, 100ul per well, spread 10 plates, and culture in an incubator (37°C, 5% CO ₂ ).

4) Screening and detection of hybridoma cells

① After cell fusion, carefully observe the growth status of the clone and whether there is contamination every day to ensure that the cells are in a normal growth state.

②After observing that the number of cells in the clonal group reaches hundreds or more (about 7 days), prepare to change the medium. Discard the medium supernatant in the fusion plate with a row gun, then add 200ul of fresh HT medium to each well, and culture in an incubator (37°C, 5% CO ₂ ).

③Two days after changing the medium, aspirate 100ul of the supernatant from each well of the 96-well plate and transfer it to an ELISA plate coated with the antigen, and mark the corresponding number. Elisa detects whether there is a specific antibody in the supernatant.

④ For the wells with positive Elisa results, observe the cells in the wells corresponding to the numbers on the plate. If there are cells, suck out the culture solution in the wells and add 200ul fresh HT medium, and culture them in an incubator (37°C, 5% CO ₂ ).

⑤ Two days after changing the medium, aspirate 100ul of the supernatant from each well of the 96-well plate and transfer it to the antigen-coated ELISA plate, mark the corresponding number, and retest by Elisa.

C. Subcloning and strain establishment

Limiting dilution method:

In the process of immunizing animals, antigens with higher purity should be selected, because an antigen theoretically has many epitopes, and the cellular immune response produced by an animal after being stimulated by exogenous antigens under normal conditions is essentially What is produced is not an antibody, but the antibody secretion of many B lymphocyte populations. The B cells targeting the target antigen epitope account for only a very small part. Therefore, the fused cells should be fully diluted in the screening to make the distribution The number of cells in each well of the culture plate is between 0 and several cells (the number of cells in 30% of the wells is 0, which can ensure that there are single cells in each well).

①Elisa re-examination results are positive wells, ready for subcloning.

②Pipe the cells in the 96-well plate with a pipette gun to make a suspension, take a small amount of the suspension and stain it with trypan blue staining solution and count it. According to the counting results, take 120 cells and add them to 10ml HT medium, and inoculate them in the pre-inoculated rearing cells respectively. Cells were placed in a 96-well plate with 100 ul per well, marked on the well plate, and cultured in an incubator (37° C., 5% CO ₂ ).

③Observe the growth status of the clones and whether there is any pollution every day to ensure that the cells grow in a normal state. When the clones in the 96-well plate grow to 1/6 of the whole well, mark the monoclonal and polyclonal, and select 24 well-grown cells for each plate. clones, numbered.

④ One day later, aspirate 100ul of the supernatant from each well of the 96-well plate and transfer it to the antigen-coated microtiter plate, mark the corresponding number, and detect the supernatant by Elisa.

⑤ For wells with positive Elisa results, select monoclonal cells with relatively high absorbance and good growth status, continue subcloning, and repeat 3-4 times until all cells in the supernatants of the wells have positive Elisa results, and strains can be established.

⑥ After the establishment of the strain, transfer the cells from the 96-well plate to the 24-well plate. After the cells are full, transfer them to the T-25 square bottle. After the cells are full, transfer them to the T-75 square bottle. When the cells are in good condition, prepare to freeze. live.

⑦Pipe the cells with an elbow pipette to make a suspension, absorb a small amount and count, calculate the total number of cells, and centrifuge at 1200rpm for 6min. ⑧ Discard the supernatant, resuspend the cells in the cryopreservation medium, divide into cryopreservation tubes (2×10 ⁶ cell/ml/tube), mark them well, and seal them with parafilm.

⑨Put the cells in a programmed cooling box and transfer them to a -80°C refrigerator.

⑩After 2 days, resuscitate one branch of cells. The next day, the microscopic examination of the cell viability > 80% proves that the cryopreservation is qualified, and transfer it to a liquid nitrogen tank within a week; if the cell viability is < 80%, it needs to be frozen again.

D. Monoclonal antibody production

1) Recovery of hybridoma cells

①Take out a cell from liquid nitrogen, put it into a 37°C water bath quickly, and shake it constantly.

② After the cells are completely thawed, wipe the cryopreservation tube with 75% alcohol and put it in an ultra-clean bench, suck out the cells and transfer them to 10ml DMEM medium, and centrifuge at 1200rpm for 5min.

③ Discard the supernatant, resuspend the pellet, mix with 5ml of complete medium, transfer to a T-25 square flask, and culture in an incubator (37°C, 5% CO ₂ ).

2) Inoculation of hybridoma cells

① Pre-inject paraffin oil into mice (0.5ml/mouse, at least 7 days after injection of paraffin oil can be used to prepare ascites).

② Collect the cells when the cells are basically covered with the bottom of the bottle and the cells are in good condition. The hybridoma cells were resuspended and counted with an elbow pipette, and centrifuged at 1200rpm for 5min.

③ Discard the supernatant, resuspend the cells with a small amount of DMEM medium, and inject intraperitoneally into paraffin mice (1-2×10 ⁶ cells per mouse).

3) Collection and preservation of ascites

①Observe the growth status of mice every day after inoculation of hybridoma cells.

②Before the mice were dying, the mice were killed by cervical dislocation, and the abdominal skin was cut with surgical scissors to expose the peritoneum, and then a small opening was cut in the peritoneum, and the ascites was sucked out with a dropper.

③ Centrifuge the ascites aspirated at 3000rpm for 30min, absorb the supernatant, aliquot and freeze at -80°C, and take a small amount of ELISA to test the titer of ascites.

4) Antibody purification

① Take 1 mL of ascitic fluid and centrifuge at 12,000 rpm for 4 minutes, take the supernatant and add 4 mL of PBS to dilute.

②Take out the protein G column (column volume 1ml), equilibrate with 10ml PBS, after the PBS dries up, load the ascites sample through the column, collect and flow through.

③Equilibrate with 10ml PBS, after the PBS is drained, add 1ml of 0.1M pH2.5 glycine solution, drain and then add 2.5ml of 0.1M pH2.5 glycine solution, collect the eluate, add 250ul 1M Tris The solution (pH8.8) was neutralized.

④ Add 10ml of PBS to balance, add 2ml of 20% ethanol after draining, and store in the column at 4°C.

The antibody eluate was dialyzed overnight with PBS, and the titer was detected by Elisa.

Example 2

A monoclonal antibody 5A10 of the present invention that can rapidly split platelet clots and inhibit vascular endothelial neogenesis binds to platelets. Centrifuge human blood samples at 1000rpm for 15min to extract platelet-rich plasma. Discard the precipitate, collect the supernatant in an anticoagulant tube, and spread 10ul of the supernatant per well in a 96-well plate. Add four kinds of monoclonal antibodies (antibody final concentration: 50ug/ml) to the 96-well plate respectively, set up 3 parallel groups for each antibody, and incubate at room temperature for 2 hours. Add FITC-labeled fluorescent secondary antibody to the 96-well plate and incubate at room temperature for 1 h. Fluorescence shift was detected by flow cytometry after the samples were filtered. Referring to Figure 2, it shows that the monoclonal antibody 5A10 can specifically bind to platelets.

Example 3

A monoclonal antibody 5A10 of the present invention that can rapidly lyse platelet clots and inhibit vascular endothelial neogenesis binds to endothelial cells. HUVECs grow to 70%-80%, discard the culture medium, wash once with PBS (Gibco, 20012-027), digest with 0.25% trypsin-EDTA (Gibco, 25200-056) at room temperature for about 1min, and gently discard the trypsin , add fresh complete medium to stop the digestion, gently blow off the adherent cells, transfer to a centrifuge tube, 1000rpm, centrifuge for 5min, discard the supernatant, wash once with PBS on ice, and resuspend in Tyrode's buffer (containing HEPES; Beyotime, ST090) Suspend cells and count. This experiment was divided into Buffer, IgG control group and antibody 1C1, 1E7, 5A10 experimental group, and three concentration gradients of 1 μg/ml, 10 μg/ml, and 100 μg/ml were set for each antibody group. Take 5×10 ⁵ cells from each part, add Buffer and each antibody according to the corresponding concentration, incubate on ice for 30 min, wash with ice Tyrode's buffer containing 0.35% BSA (Bovine Serum Albumin; Beyotime, ST023) for 2~ 3 times, 4°C, 1000rpm, 5min centrifugation. Add FITC (fluorescein isothiocyanate, Fluorescein Isothiocyanate) labeled goat anti-mouse IgG (Beyotime, A0568) at a dilution ratio of 1:500, incubate on ice in the dark for 30 min, and wash 1-2 times with ice Tyrode's buffer. Add 500 μl ice Tyrode's buffer to each portion to prepare 1×10 ⁶ /ml cell final solution. Filter with 300-mesh nylon membrane (MultiSciences, N3002), add 150-200 μl/well into a 96-well plate, and set 2-3 replicate holes for each portion. The 96-well plate was put into a flow cytometer (Guava, easyCyte 6HT-2L) for analysis, and 1×10 ⁴ cells were collected in each well. Flow cytometry software M guavasoft2.7 built-in function processing results. Referring to Figure 3, it shows that the monoclonal antibody 5A10 can specifically bind to human umbilical vein endothelial cells (HUVEC).

Example 4

A monoclonal antibody 5A10 of the present invention that can rapidly split platelet clots and inhibit vascular endothelial neogenesis induces platelet clots to depolymerize. Human platelets were added to 96-well culture plate at 1×10 ³ per well, then 100 mg/mL fibrinogen and 10 μM ADP were added and incubated at 37°C for 30 min to induce platelet clot formation. After centrifugation at 1000rpm for 15min, the supernatant was discarded. Add 5A10 100 mg/mL and irrelevant control reagents, and at different time points, count the number of platelet clots per 1000 platelets under a microscope (that is, the disaggregation constant of platelet clots). Referring to FIG. 4 , it is shown that monoclonal antibody 5A10 induces platelet clot disaggregation with increasing time.

Example 5

A monoclonal antibody 5A10 capable of rapidly cleaving platelet clots and inhibiting vascular endothelial neogenesis of the present invention inhibits HUVEC proliferation. Time-lapse determination: 200 μl/well of 0.5% gelatin was spread on a 96-well plate in advance, and placed overnight in a 37° C., 5% CO ₂ cell culture incubator. Take logarithmic phase cells, digest, count, adjust cell density, and prepare cell suspension. In this experiment, zero wells were set (only complete medium was added without adding cells), and they were divided into Buffer, IgG control group and antibody 1C1, 1E7, 5A10 experimental groups, and each group was set with 5 replicate wells. 180 μl/well of the above cell suspension was added to a 96-well plate (5000/well), and PBS was added to the surrounding wells to seal the plate. After overnight culture, the cells adhered to the wall. Antibodies IgG, 1C1, 1E7, and 5A10 were added at a final concentration of 100 μg/ml, and PBS was added to the Buffer group. Add 20μl/well 5mg/ml MTT (Methylthiazolyldiphenyl-tetrazolium bromide; Beyotime, ST316) at 1 day, 2 days, and 3 days after adding the antibody, and incubate in a 5% CO ₂ incubator at 37°C in the dark 4h, discard the supernatant, add 150μl/well DMSO (Sigma, D2650), place at 37°C for 10min and shake until the formazan crystals are completely dissolved, blow the air bubbles in the holes with a hair dryer, and use a microplate reader (Molecular Devices, SpectraMax M5) at 550nm Determine the _OD550 value. The calculation of the inhibition rate IR of the result analysis is according to the following formula:

Referring to FIG. 5 , it shows that the anti-GPIIIa49-57 monoclonal antibody 5A10 can significantly inhibit the proliferation of human umbilical vein endothelial cells (HUVEC) compared with the control antibody.

Example 6

A monoclonal antibody 5A10 of the present invention, which can rapidly lyse platelet clots and inhibit vascular endothelial neogenesis, induces HUVEC apoptosis. The cells were recovered and subcultured once, and after 2 to 3 days, they grew to 70% to 80%, digested and counted, and took an equal amount of cells to 3 culture dishes. Buffer, IgG (100 μg/ml) and antibody 5A10 (100 μg/ml) were added to the cells in the same growth state to pretreat the cells. After 48 hours of antibody treatment, the cells in the logarithmic phase were collected, the supernatant and the suspended cells in it were collected into a centrifuge tube, and the cells were digested with 0.25% trypsin (Gibco, 15050-065) without EDTA, and the latter was mixed with the corresponding supernatant medium Combine, centrifuge at 4°C, 1200rpm for 3min, and discard the supernatant. Wash the cells 1-2 times with pre-cooled PBS and count them, centrifuge at 1200 rpm at 4°C for 3 min, discard the supernatant, and place the cells on ice. In this experiment, FITC-Annexin V Apoptosis Detection Kit (DOJINDO, AD10) was used to detect cell apoptosis, and further optimized and refined according to the needs of the experiment: 10×Annexin V Binding Buffer was used in advance with Milli-Q Water (Merk, Merk -Milli-QAdvantage) was diluted 10 times. Add pre-cooled 1×Annexin V Binding Buffer to prepare a single cell suspension, and the cell density is 1×10 ⁶ /ml (above 500 μl). For each sample, take 100 μl of the above single-cell suspension to a new 1.5ml EP tube; in addition, for each sample, take an equal volume of the above-mentioned single-cell suspension and mix it into 100 μl/part (3 parts) for the preparation of adjustment samples. Add 5 μl FITC-Annexin V conjugate to each sample, mix gently, and incubate on ice in the dark for 15 minutes; then add 400 μl pre-cooled 1×Annexin V Binding Buffer, and mix gently; immediately add 5 μl PI solution, mix gently Mix well, and place on ice in the dark. In addition, 1 part of the adjustment sample was unstained cells, 1 part was FITC-Annexin V single-stained cells, and 1 part was PI single-stained cells. Other operations were the same as the staining steps of the test samples. Filter through a 300-mesh nylon membrane, add 150-200 μl/well to a 96-well plate, set 2-3 multiple wells for each sample, and set 1 well for each calibration sample. Place in the flow cytometer for analysis within 5 minutes, collect 1×10 ⁴ cells per well, and complete the detection within 1 hour at most. The flow cytometer needs to be washed 15-20 minutes in advance before it can be used for detection. Flow cytometry software M guavasoft2.7 built-in function processing results. Referring to FIG. 6 , monoclonal antibody 5A10 can induce apoptosis of human umbilical vein endothelial cells (HUVEC).

Example 7

A monoclonal antibody 5A10 capable of rapidly cleaving platelet clots and inhibiting vascular endothelial neogenesis of the present invention inhibits HUVEC migration. The cells were recovered and subcultured once, and grew to 70% to 80% after 2 to 3 days, digested and counted, and took an equal amount of cells to five 6cm culture dishes (160,000/dish). After the cells adhered to the wall overnight, discarded In the original culture medium, Buffer, IgG (100 μg/ml) and antibody 5A10 (1 μg/ml, 10 μg/ml, 100 μg/ml) were added to 5 dishes of cells with the same growth status to pretreat the cells. After 48 hours of antibody treatment, the complete antibody-containing medium was discarded, washed 1-2 times with PBS, replaced with basal medium (ECM+P/S, no FBS and no ECGS), and starved for 4-6 hours. Take the logarithmic phase cells, digest the cells with 0.25% trypsin-EDTA, aspirate and discard the trypsin (the cells are almost not discarded), add fresh complete medium to terminate the digestion (ensure that the serum is sufficient to terminate the digestion), 1000rpm, 5min, aspirate the supernatant, Wash once with PBS, resuspend in basal medium, count, take a certain amount of cells and add basal medium to adjust the cell density to 2.5×10 ⁵ /ml. Transwell chamber (8 μm; Costar, 3422) was pre-hydrated and balanced, added 600 μl of liquid to the lower chamber, put it into the small chamber (to ensure no air bubbles), added 100 μl of liquid to the upper chamber, added basal medium to both upper and lower chambers, and kept overnight at 37°C in 5% CO ₂ (at least 1h) place it, discard the culture medium and set aside. Add 600 μl of complete medium (ECM+5% FBS+1% ECGS+P/S) to the lower chamber and put it into the small chamber (make sure there are no air bubbles), and add 100 μl of the above cell suspension (2.5×10 ⁴ /small chamber) to the upper chamber. Set up 3 parallel chambers. Incubate for 4 h at 37°C in a 5% CO ₂ cell incubator. Fix with methanol at room temperature for 30 min, and wash with sterile PBS 2 to 3 times. Stain with 0.1% crystal violet (Bogoo, PT007) at room temperature for 30 minutes, and wash with sterile PBS 2 to 3 times. Carefully wipe off the non-migrated cells on the upper side of the chamber membrane with a wet cotton swab, and use continuous and uniform force to avoid membrane rupture, especially the upper edge cells of the chamber membrane, which will significantly affect the results and need to be wiped off. The chambers were left to dry overnight. Inverted fluorescence microscope (Olympus, DP80, IX73) selects bright field and 100× magnification, and takes 9-10 random fields of view to take pictures. Decolorize with 10% acetic acid (glacial acetic acid: distilled water = 1:9) on a shaker for 10 min, transfer the eluate to a 96-well plate, and read the OD ₅₉₅ value at 595 nm with a microplate reader. Referring to FIG. 7 , monoclonal antibody 5A10 inhibits the migration of human umbilical vein endothelial cells (HUVEC) in a dose-dependent manner.

Example 8

The monoclonal antibody 5A10 of the present invention, which can rapidly lyse platelet clots and inhibit vascular endothelial neogenesis, inhibits HUVEC angiogenesis. BME-RGF (Trevigen, 3433-005-01) was placed in an ice-water bath at 4°C in advance overnight; the BME-RGF solution had to be placed on ice all the time during the experiment, and if it was higher than 8°C, it would solidify and polymerize. Pre-cool 200 μl pipette tips and 96-well plates at 4°C. In this experiment, the in vitro angiogenesis assay kit (Trevigen, 3470-096-K) was used to measure the angiogenesis of HUVECs, and further optimized and refined according to the needs of the experiment: centrifuge 50 μg Calcein-AM powder at 12000 rpm for 1 min (Trevigen, 4892-010-01 ), open the lid, add 25 μl sterile DMSO (25 μl stock solution; 2 μg/μl, 2 mM), mix up and down with a pipette tip, and store at -20°C; re-dilute before each experiment, add 4 μl stock solution at 1:1000 4ml PBS (4ml final solution; 2μΜ). Add 2 μl of 10 mM Negative CT (Sulforaphane; Trevigen, 3470-096-02) to 98 μl of basal medium (100 μl final solution; 200 μM) at a ratio of 1:50; it needs to be prepared again before each experiment. In the ultra-clean bench, put the BME solution on ice, balance the pre-cooled 96-well plate on the ultra-clean bench, and put 60 μl/well of BME in the center of the well into the 96-well plate with the pre-cooled pipette tip to ensure that it is evenly covered and free of air bubbles , Seal the plate with PBS in the wells around the glue. Place at 37°C for 30-60min. Take logarithmic phase cells, digest and collect, wash once with PBS, add complete medium to resuspend into single cell suspension, and count accurately. Take a certain volume of mixed cell solution, add complete medium to adjust the final cell density to 100,000/ml. Add 1 μg/ml VEGF (Recombinant Murine VEGF ₁₆₅ ; PEPROTECH, 450-32) to the final cell solution at a ratio of 1:200 to a final concentration of about 5 ng/ml. Mix the final solution of 100,000/ml cells, divide into equal volumes, and add corresponding Buffer, Negative CT (20 μM), IgG (100 μg/ml), 5A10 (1 μg/ml, 10 μg/ml, 100 μg/ml). The cell suspension was mixed, and 100 μl/well was slowly added to a 96-well plate at a fixed position along the same direction of the well wall (10,000 per well), and each group was set up with 4 replicate wells. Incubate for 6 hours at 37°C in a 5% CO ₂ cell incubator. Aspirate and discard the culture medium in the well, wash once with 150 μl/well PBS, add 100 μl/well 2 μM Calcein-AM, incubate at 37°C, 5% CO ₂ for 30 min, carefully aspirate Calcein-AM, add 100 μl/well PBS. Invert the fluorescent microscope, select the Blue2 channel (blue light excites green light) and a 40× field of view, and take pictures. Quantitative indicators such as the total length of tubules and the ratio of cell coverage area were counted by Image J software, and the results were analyzed. Referring to Figure 8, the monoclonal antibody 5A10 inhibited the formation of blood vessels by endothelial cells in a dose-dependent manner.

The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages conceivable by those skilled in the art are all included in the present invention, and the appended claims are the protection scope.

Claims (4)

1. An anti-platelet integrin glycoprotein IIIa monoclonal antibody 5A10 is characterized in that this monoclonal antibody 5A10 is by using platelet integrin GPIIIa49-57 fragment immunization integrin GPIIIa deficient BALB/c mice, and using hybridoma The heavy chain of the antibody is IgG ₁ type, the light chain is κ type, and the molecular weight of the antibody is 150 KDa. 2. a preparation method of anti-platelet integrin glycoprotein IIIa monoclonal antibody 5A10 according to claim 1, characterized in that the method comprises the following steps: a) The chemically synthesized platelet integrin GPIIIa49-57 small peptide (CAPESIEFP fragment) was coupled with keyhole limpet hemocyanin (KLH), and BALB/c mice deficient in integrin GPIIIa were immunized by subcutaneous multi-point immunization; the antigen was used in PBS Dilute to 0.3 mg/mL, mix 1000 uL with 1000 uL Freund's complete adjuvant or Freund's incomplete adjuvant, emulsify, subcutaneously immunize at multiple points; immunize once every 2 weeks, and immunize 4 times in total; after the last immunization, Orbital venous blood was collected from the mice, serum was collected, and the titer of the mouse serum was detected by Elisa method; qualified mice that were positive in the Elisa test were selected, and 50 ug of each antigen was directly injected intraperitoneally into the qualified mice, and cell fusion was performed three days later; b) Splenocytes from immunized mice were fused with myeloma cells (SP2/0 cells), screened by HAT selective medium, and subcloned to obtain a hybridoma cell line with anti-platelet integrin GPIIIa49-57 monoclonal antibody, named: HYD -5A10; wherein, H of HAT: hypoxanthine A: aminopterin T: thymine nucleotide; c) Hybridoma cell line HYD-5A10 was inoculated into mice that had been injected intraperitoneally with paraffin oil. After inoculation of HYD-5A10 cells, the growth status of the mice was observed every day. The ascites was aspirated and purified by a protein G column to obtain the anti-platelet integrin glycoprotein IIIa monoclonal antibody 5A10; wherein, the inoculum volume of HYD-5A10 was 1-2×10 ⁶ cells/mouse. 3. The application of the anti-platelet integrin glycoprotein IIIa monoclonal antibody 5A10 according to claim 1 in the preparation of medicaments for treating arterial thrombosis diseases. 4. The application of the anti-platelet integrin glycoprotein IIIa monoclonal antibody 5A10 according to claim 1 in the preparation of anti-tumor drugs.