A method for preparing branched polypeptides that simulate the antigenicity of CD31 linear molecules

Abstract

The present invention belongs to the technical field of monoclonal antibody preparation, and specifically relates to a method for preparing a branched polypeptide that simulates the antigenicity of CD31 linear molecules. The method comprises the following steps: step 1. experimental material preparation; step 2. experimental instrument preparation; step 3. amino acid activation; step 4. amino acid connection to resin; step 5. removal of Fmoc protecting group of amino acid; step 6. peptide resin cleavage; step 7. polypeptide separation; step 8. polypeptide separation, purification and analysis. The invention successfully prepares monoclonal antibodies using branched peptide chains instead of linear peptide chains, which has production significance. This method reduces costs, speeds up preparation, and improves antibody quality. Peptide synthesis only takes a short time. The prepared monoclonal antibodies can be used for WB and IHC experiments, meeting the needs of different antigenic determinants. In short, the monoclonal antibodies prepared by branched peptide chains have the characteristics of precise positioning, low cost, high efficiency and versatility.

Description

Technical Field

[0001] The invention belongs to the technical field of monoclonal antibody preparation, and particularly relates to a method for preparing a branched polypeptide that simulates the antigenicity of CD31 linear molecules. Background Art

[0002] CD31 is also known as platelet endothelial cell adhesion molecule-1 (PECAM-1 / CD31), with a molecular weight of 130 kDa. Its structure belongs to the immunoglobulin superfamily and plays an important role in the process of clearing aging neutrophils in the body. CD31 exists on the surface of platelets, neutrophils, monocytes and certain types of T cells, as well as in the tight junctions between endothelial cells. Literature shows that CD31 is involved in the migration of leukocytes, angiogenesis and activation of integrins. CD-31 is usually located in vascular endothelial cells, platelets, macrophages and Kupffer cells, granulocytes, T cells, NK cells, lymphocytes, megakaryocytes, osteoclasts, and neutrophils. CD31 expression is also found in some tumors, such as epithelioid hemangioendothelioma, epithelioid sarcoma-like hemangioendothelioma, other vascular tumors, histiocytic malignancies, and plasma cells, but is rarely found in sarcomas, such as Kaposi's sarcoma. In immunohistochemistry, CD31 is mainly used to evaluate tumor angiogenesis and to prove the presence of endothelial cell tissue. Highly proliferative blood vessels mean that the tumor is more malignant.

[0003] CD31 protein is a popular target for scientific research, and there is a huge market demand for the development of high-quality anti-CD31 antibodies. However, the quality of CD31 antibodies on the market varies greatly. The existing problems are: 1. The antibodies used for flow cytometry, immunohistochemistry and immunoblotting cannot be unified, so there are many types of antibodies with the same name and different performances, which causes great selection difficulties for customers. 2. The staining effect is not good, which is manifested in the following problems: in immunoblotting detection, the color bands are not single, the bands are blurred, and the position is incorrect. In immunohistochemical staining, the antigen is not clearly located in the cell, and the foreground and background are not obvious enough. There are many defects. 3. Some antibodies have poor binding specificity. The CD31 antibody should be combined with other molecules with high homology. For this reason, we plan to develop a monoclonal antibody with high specificity and good versatility that can be used for both immunohistochemical staining and immunoblotting staining. In the development of monoclonal antibodies, the selection of antigen peptides is a key factor in determining the properties of antibodies.

[0004] Peptides have weak immunogenicity. It is difficult to induce an effective immune response using one antigenic determinant. Using too long a peptide will also cause unclear immune epitopes. If the same linear epitope is repeated, new combinations of amino acids will be formed, forming non-specific antibodies. To overcome this situation, the traditional process is to use keyhole limpet protein or bovine serum albumin to enhance the immune response. However, keyhole limpet protein and bovine serum albumin themselves can induce a large number of non-specific binding antibodies, which reduces the quality of antibody preparation and increases the difficulty of antibody selection. For this reason, we propose a method for preparing branched polypeptides that simulate the antigenicity of CD31 linear molecules to solve the above problems. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a branched polypeptide that simulates the antigenicity of CD31 linear molecules. The branched peptide chain can be used instead of the linear peptide chain, and the monoclonal antibody is successfully prepared. The antigen epitope is accurately located, and the mechanism is clear, which is more popular among scientific researchers and reduces the cost. The short peptide is composed of several amino acids and can be directly synthesized by chemical methods. If the complete or partial protein is synthesized by synthetic methods, the cost is high due to the length. The expression by genetic recombination is also costly, which speeds up the preparation speed. The polypeptide synthesis only takes a short time, and the antibody quality is improved. Since the length of the amino acid chain is shortened, it can be used for WB immunohistochemical staining and flow cytometry identification at the same time: since polyclonal antibodies are used, tens of thousands of antibodies can be produced simultaneously for different antigenic determinants of the polypeptide. Among these antibodies, some are for linear antigens, and some are for nonlinear antigens with secondary structures, so the produced polyclonal antibodies can be used for WB and IHC.

[0006] The technical solution adopted by the present invention is as follows:

[0007] A method for preparing a branched polypeptide that simulates the antigenicity of a CD31 linear molecule, the method comprising the following steps:

[0008] Step 1. Preparation of experimental materials;

[0009] Step 2. Prepare experimental instruments;

[0010] Step 3. Amino acid activation;

[0011] Step 4. Connecting amino acids to the resin;

[0012] Step 5. Removal of the Fmoc protecting group of the amino acid;

[0013] Step 6. cleavage of the peptide resin;

[0014] Step 7. Separation of polypeptides;

[0015] Step 8. Isolation, purification and analysis of peptides.

[0016] In a preferred embodiment, the experimental materials include P-hydroxymethylphenoxymethyl polyethylene resin, Wang resin, Fmoc-AA, NMP nitrogen methyl pyrrolidone, DCM dichloromethane, MeoH methanol, Piperidine piperidine, DMAP dimethylaminopyridine, HOBT hydroxybenzotriazole, DCCN, N'-dicyclohexylcarbodiimide, TFA trifluoroacetic acid, EDT1,2-ethanedithiol, thioanisole, crystalline phenol, and acetonitrile.

[0017] In a preferred embodiment, the experimental instruments include an automatic polypeptide synthesizer, a rotary evaporator, a high performance liquid chromatograph, a freeze dryer, a vacuum circulating water pump, and a centrifuge.

[0018] In a preferred embodiment, the amino acid activation is to connect the carboxyl group of the glycine at the carboxyl terminal of the compound to an insoluble polymer compound carrier HMP resin in the form of a covalent bond.

[0019] In a preferred embodiment, the amino acid is connected to the resin by removing the protecting groups of the α-amino group and the side chain amino group after the second amino acid at the carboxyl end, ie, lysine, is synthesized to expose two free amino groups and couple two identical lysines.

[0020] In a preferred embodiment, the Fmoc protecting group of the amino acid is removed by simultaneously removing the protecting groups of the α-amino group and the side chain amino group of the two lysines. At this time, there are four free amino groups, which are then coupled with an excess of activated carboxyl components. In this way, four lysines are coupled simultaneously, and the eight amino protecting groups on the four lysines are then removed. This is an eight-branched lysine backbone, and eight identical polypeptides are then connected to this backbone at the same time, VMAMVEHSGNYTC-GSK, to obtain a polypeptide derivative with complete immunogenic biological activity: (VMAMVEHSGNYTC-GSK)8(Kys)4-(Lys)2-Lys-Gly.

[0021] In a preferred embodiment, the cleavage of the peptide resin is to cut the polypeptide from the peptide resin with TFA: use EDT, thioanisole, and H2O as scavengers, react at room temperature for 3.0 hours, remove the cleavage reagent, and then extract with ether to obtain a crude (VMAMVEHSGNYTC-GSK)8(Kys)4-(Lys)2-Lys-Gly polypeptide compound.

[0022] In a preferred embodiment, the polypeptide is separated by reacting the peptide resin with a scavenger mixed with trifluoroacetic acid, 1,2-ethanedithiol, thioanisole and water to separate the CD31 or comparative example polypeptide fragment from the peptide resin; after the reaction, the resin is filtered out and the scavenger is removed by reduced pressure distillation; the CD31 or comparative example polypeptide fragment is dissolved in water and extracted with ether to obtain a crude CD31 or comparative example polypeptide compound.

[0023] In a preferred embodiment, the separation, purification and analysis of the polypeptide is to separate and purify the crude CD31 or comparative polypeptide compound by high performance liquid chromatography, and the specific operating conditions are: chromatographic column: C1825×250mm, chromatograph: Waters600, mobile phase: A-0.1% TFA, H2O, B-0.1% TFA in 60% acetonitrile, detection wavelength: 214nm, flow rate: 10ml / min, elution gradient from 20-60%, thereby, a polypeptide with a purity of>90% can be obtained to meet the immune requirements.

[0024] In a preferred embodiment, the separation, purification and analysis of the polypeptide further involves high performance liquid chromatography analysis of the separated and purified CD31 polypeptide, and the analysis conditions are: chromatographic column: C18 4.6×150 mm, mobile phase: A-0.1% TFA, H2O, B-0.1% TFA, acetonitrile, detection wavelength: 214 nm, flow rate: 1 ml / min, elution gradient from 0-60%.

[0025] The technical effects achieved by the present invention are:

[0026] Using branched peptide chains instead of linear peptide chains, monoclonal antibodies were successfully prepared. The antigen epitope was precisely located, and the mechanism was clear, which was more popular among scientific researchers and reduced costs. Short peptides are composed of several amino acids and can be directly synthesized by chemical methods. If the complete or partial protein is synthesized by synthetic methods, the cost is high due to its long length. The expression by genetic recombination also requires an expression system, which also causes high costs. Peptide synthesis only takes a short time, which speeds up the preparation speed and improves the quality of antibodies. Due to the shortened length of the amino acid chain, it can be used for WB immunohistochemical staining and flow cytometry identification at the same time: Because the branched antigen with better immunogenicity is used, the antibodies produced have high affinity and good specificity, and have been proven to be used for WB and IHC. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a schematic diagram of a method for preparing a branched polypeptide that simulates the antigenicity of a CD31 linear molecule of the present invention;

[0028] Figure 2 It is a schematic diagram of connecting amino acids to a resin in a method for preparing a branched polypeptide simulating the antigenicity of a CD31 linear molecule of the present invention;

[0029] Figure 3 It is a schematic diagram of a method for preparing a branched polypeptide simulating the antigenicity of a CD31 linear molecule of the present invention, wherein the Fmoc protecting group of the amino acid is removed;

[0030] Figure 4 It is a schematic diagram of amino acid activation of a method for preparing a branched polypeptide simulating the antigenicity of a CD31 linear molecule of the present invention;

[0031] Figure 5 It is a schematic diagram of amino acid coupling of a method for preparing a branched polypeptide simulating the antigenicity of a CD31 linear molecule of the present invention;

[0032] Figure 6 It is a schematic diagram of the amino acid branching structure of a method for preparing a branched polypeptide simulating the antigenicity of a CD31 linear molecule of the present invention. DETAILED DESCRIPTION

[0033] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the accompanying drawings.

[0034] Embodiment 1

[0035] See also Figure 1-6 As shown, the present invention provides a method for preparing a branched polypeptide that simulates the antigenicity of a CD31 linear molecule. The method for preparing a branched polypeptide comprises the following steps:

[0036] Step 1. Preparation of experimental materials;

[0037] Step 2. Prepare experimental instruments;

[0038] Step 3. Amino acid activation;

[0039] Step 4. Connecting amino acids to the resin;

[0040] Step 5. Removal of the Fmoc protecting group of the amino acid;

[0041] Step 6. cleavage of the peptide resin;

[0042] Step 7. Separation of polypeptides;

[0043] Step 8. Isolation, purification and analysis of peptides.

[0044] The experimental materials include P-hydroxymethylphenoxymethyl polyvinyl resin, Wang resin, Fmoc-AA, NMP nitrogen methyl pyrrolidone, DCM dichloromethane, MeoH methanol, Piperidine piperidine, DMAP dimethylaminopyridine, HOBT hydroxybenzotriazole, DCCN, N'-dicyclohexylcarbodiimide, TFA trifluoroacetic acid, EDT1,2-ethanedithiol, thioanisole, crystalline phenol, acetonitrile;

[0045] The experimental equipment includes an automatic peptide synthesizer, a rotary evaporator, a high performance liquid chromatograph, a freeze dryer, a vacuum circulating water pump, and a centrifuge. The amino acid activation is to connect the carboxyl group of the glycine at the carboxyl terminal of the compound to an insoluble polymer compound carrier HMP resin in the form of a covalent bond. The amino acid is connected to the resin by removing the protecting groups of the α-amino group and the side chain amino group at the same time after the second amino acid at the carboxyl terminal, i.e., lysine, is synthesized to expose two free amino groups, and two identical lysines are coupled.

[0046] The Fmoc protecting group of amino acids is removed by removing the protecting groups of the α-amino group and the side chain amino group of the two lysines at the same time. At this time, there are four free amino groups, which are then coupled with excess activated carboxyl components, so that four lysines are coupled at the same time. The eight amino protecting groups on the four lysines are then removed, and this is an eight-branched lysine skeleton. Eight identical polypeptides are then connected to this skeleton at the same time, VMAMVEHSGNYTC-GSK, to obtain a polypeptide derivative with complete immunogenic biological activity: (VMAMVEHSGNYTC-GSK)8(Kys)4-(Lys)2-Lys-Gly;

[0047] The peptide resin was cleaved by using TFA to cut the polypeptide from the peptide resin: EDT, thioanisole, and H2O were used as scavengers, reacted at room temperature for 3.0 hours, the cleavage reagent was removed, and then extracted with ether to obtain a crude product of (VMAMVEHSGNYTC-GSK)8(Kys)4-(Lys)2-Lys-Gly polypeptide compound;

[0048] The separation of the polypeptide comprises reacting a scavenger mixed with 1,2-ethanedithiol, thioanisole and water with a peptide resin to separate the CD31 or comparative polypeptide fragment from the peptide resin; filtering off the resin after the reaction, and removing the scavenger by vacuum distillation; dissolving the CD31 or comparative polypeptide fragment in water, and extracting with ether to obtain a crude CD31 or comparative polypeptide compound;

[0049] Separation, purification and analysis of polypeptides: The crude CD31 or comparative polypeptide compound was separated and purified by high performance liquid chromatography. The specific operating conditions were as follows: chromatographic column: C18 25×250 mm, chromatograph: Waters 600, mobile phase: A-0.1% TFA, H2O, B-0.1% TFA in 60% acetonitrile, detection wavelength: 214 nm, flow rate: 10 ml / min, elution gradient from 20-60%, thereby, a polypeptide with a purity of >90% was obtained to meet the immune requirements. Separation, purification and analysis of polypeptides: The separated and purified CD31 polypeptide was further subjected to high performance liquid chromatography analysis, and the analysis conditions were as follows: chromatographic column: C18 4.6×150 mm, mobile phase: A-0.1% TFA, H2O, B-0.1% TFA, acetonitrile, detection wavelength: 214 nm, flow rate: 1 ml / min, elution gradient from 0-60%;

[0050] The sequence of each branch in the amino acid branch structure is as follows: Serine-Leucine-Lysine-Aspartic Acid-Tryptophan-Threonine-Valine-Lysine, the sequence of each branch in the amino acid branch structure is as follows: ending with lysine and constructed with an acidic amino acid at the 5th position, amino acid sequence: Asparagine-Serine-Arginine-Asparagine-Glutamine-Asparagine-Phenylalanine-Valine-Isoleucine-Leucine-Lysine, asparagine is densely used at positions 1, 4, and 6 to regulate each peptide chain of the branched peptide to enhance antigenicity, amino acid sequence: Histidine-Phenylalanine-Leucine-Asparagine-Serine-Aspartic Acid-Valine-Glutamine-Tyrosine-Threonine-Lysine, starting with a basic amino acid, combined with asparagine at positions 4 and 6 to enhance antigenicity.

[0051] Embodiment 2

[0052] Preparation and detection of CD31 monoclonal antibody

[0053] Immunization of animals

[0054] Take 8-12 week old Balb / c mice of the same lineage as myeloma cells, fully emulsify the antigen containing branched polypeptide with an equal amount of Freund's complete adjuvant, and inject it into the mouse's abdominal cavity. Emulsify the antigen with Freund's incomplete adjuvant every 2-4 weeks, and mostly use subcutaneous multiple injections. About one week after the second immunization, blood is collected from the tail vein to test the antibody titer. For mice that meet the requirements, 20μg / 100μL of antigen solution is directly injected into the spleen to strengthen the immunization. After 3 days, the spleen is taken for cell fusion.

[0055] 1. Myeloma cell resuscitation

[0056] Take out the frozen myeloma cells from the liquid nitrogen tank and immediately put them in a 37℃ water bath. After thawing, centrifuge at 1000r / min for 5min. Transfer the precipitated cells into a cell culture bottle, add DMEM complete culture medium, and culture in a CO2 incubator.

[0057] 2. Myeloma cell culture

[0058] Myeloma cells (Sp2 / 0) were cultured in complete culture medium containing 10% to 15% calf serum, and subcultured or expanded every 3 to 4 days. Then, cells in the logarithmic growth phase with vigorous growth and good morphology were selected for fusion.

[0059] 3. Cultivation of Feeder Cells

[0060] Take 3-4 Balb / c mice, kill them by cervical dislocation, and soak them in 75% ethanol for disinfection. Fix the mice on a wax tray with their abdomen facing up. Use sterile scissors to cut a small hole in the abdominal skin to expose the abdominal muscles. Use a sterile syringe to inject 5mL of DMEM culture medium into the mouse's abdominal cavity, and gently massage its abdomen for about 1-2 minutes. Use a syringe to extract the intraperitoneal fluid and place it in a pre-cooled 50mL centrifuge tube. Centrifuge at 1000rpm for 10 minutes, discard the supernatant, suspend the cells in 20mL of HAT culture medium, mix well, and drop the cell suspension into the small wells of a 96-well culture plate, 100μL per well, and culture in an incubator with 5%-7% CO2, 37℃, and saturated humidity.

[0061] 4. Selection and culture of hybridoma cells:

[0062] On the first day after cell fusion, add 100 μL of HAT culture medium to each well, and replace half of the HAT culture medium every 2 to 3 days. Stop using HAT culture medium on the 7th to 10th day and use HT culture medium instead. Aspirate the culture medium for specific antibody detection to determine which well contains hybridoma cells that secrete specific antibodies.

[0063] 5. Detection of specific antibodies

[0064] Add the antigen diluted to an appropriate concentration to each well of a 96-well plate, 100 μL per well, and place at 4°C for overnight adsorption. The next day, shake off the antigen solution in the well and wash three times with PBST washing solution. Add 200 μL of 0.01M pH 7.2 phosphate buffer containing 10% bovine serum albumin to each well and place at 37°C for 1 hour to close the gaps on the well wall and prevent non-specific binding. Wash again with phosphate buffer 2 to 3 times. Add 100 μL of the supernatant to be tested to each well and place it in a humidified box at 37°C for 1 hour. At the same time, set up negative and positive controls. After washing three times with washing solution, add 100 μL of HRP-labeled anti-mouse IgG secondary antibody diluted appropriately with diluent to each well and place it in a humidified box at 37°C for 1 hour. Shake off the excess enzyme-labeled secondary antibody, wash five times with washing solution, and then wash twice with distilled water. Add 100 μL of freshly prepared substrate TMB to each well and place it in the dark at room temperature for 5 to 30 minutes. When the positive control well turns blue, add 100 μL of 2 mol / L sulfuric acid to terminate the reaction. Measure the results using an enzyme-labeled reader.

[0065] 6. Inoculate hybridoma cells into the peritoneal cavity of syngeneic mice or nude mice. Specific steps:

[0066] (1) Before inoculation of hybridoma cells, mice (Balb / c mice) were intraperitoneally injected with 0.5 mL of pristane.

[0067] (2) Then 5×105-106 hybridoma cells were injected intraperitoneally into each mouse.

[0068] (3) About 7 to 10 days after inoculation of hybridoma cells, the abdomen of the mouse swells significantly. Disinfect its abdomen with 75% ethanol and pierce the sterilized injection needle (No. 7) into the lower abdomen of the mouse. Allow the ascites to drip into the test tube and collect the ascites 2 to 3 times.

[0069] (4) Centrifuge the ascites at 1500 rpm for 10 min, aspirate the supernatant, add 0.02% sodium azide, and store in aliquots at -20°C.

[0070] In the present invention, a branched peptide chain is used instead of a linear peptide chain to successfully prepare a monoclonal antibody. The antigen epitope is precisely located, and the mechanism is clear, which is more popular among scientific researchers and reduces costs. A short peptide is composed of several amino acids and can be directly synthesized by chemical methods. If a complete or partial protein is synthesized by a synthetic method, the cost is high due to its length. Expression by genetic recombination is also costly, which speeds up the preparation speed. It only takes a short time to synthesize the polypeptide, which improves the antibody quality. Since the length of the amino acid chain is shortened, it can be used for WB immunohistochemical staining and flow cytometry identification at the same time: since polyclonal antibodies are used, tens of thousands of antibodies can be produced simultaneously for different antigenic determinants of polypeptides. Some of these antibodies are directed against linear antigens, and some are directed against nonlinear antigens with secondary structures, so the produced polyclonal antibodies can be used for WB as well as IHC.

[0071] The above is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention. The structures, devices and operating methods not specifically described and explained in the present invention shall be implemented according to the conventional means in the art unless otherwise specified and limited.

Claims

1. A method for preparing a branched polypeptide that simulates the antigenicity of a CD31 linear molecule, characterized in that: The method for preparing the branched polypeptide comprises the following steps: Step 1. Preparation of experimental materials; Step 2. Prepare experimental instruments; Step 3. Amino acid activation; Step 4. Connecting amino acids to the resin; Step 5. Removal of the Fmoc protecting group of the amino acid; Step 6. cleavage of the peptide resin; Step 7. Separation of polypeptides; Step 8. Isolation, purification and analysis of peptides; The experimental materials include P-hydroxymethylphenoxymethyl polyvinyl resin, Wang resin, Fmoc-AA, NMP nitrogen methyl pyrrolidone, DCM dichloromethane, MeoH methanol, Piperidine piperidine, DMAP dimethylaminopyridine, HOBT hydroxybenzotriazole, DCCN, N'-dicyclohexylcarbodiimide, TFA trifluoroacetic acid, EDT1,2-ethanedithiol, thioanisole, crystalline phenol, acetonitrile; The experimental instruments include an automatic peptide synthesizer, a rotary evaporator, a high performance liquid chromatograph, a freeze dryer, a vacuum circulating water pump, and a centrifuge; The amino acid activation is to connect the carboxyl group of the glycine at the carboxyl terminal of the compound to an insoluble polymer compound carrier HMP resin in the form of a covalent bond; The method of coupling the amino acid to the resin is to remove the protecting groups of the α-amino group and the side chain amino group of the second amino acid at the carboxyl end, i.e., lysine, at the same time after the synthesis of the second amino acid at the carboxyl end, thereby exposing two free amino groups and coupling two identical lysines; The Fmoc protecting group of the amino acid is removed by removing the protecting groups of the α-amino group and the side chain amino group of the two lysines at the same time, so that there are four free amino groups, which are then coupled with excess activated carboxyl components, so that four lysines are coupled at the same time, and then the eight amino protecting groups on the four lysines are removed, and this is an eight-branched lysine skeleton, and eight identical polypeptides are connected to this skeleton at the same time, VMAMVEHSGNYTC-GSK, so that a polypeptide derivative with complete immunogenic biological activity is obtained: (VMAMVEHSGNYTC-GSK)8(Lys)4-(Lys)2-Lys-Gly; The cleavage of the peptide resin is to cut the polypeptide from the peptide resin with TFA: use EDT, thioanisole, and H2O as scavengers, react at room temperature for 3.0 hours, remove the cutting reagent, and then extract with ether to obtain a crude product of the (VMAMVEHSGNYTC-GSK)8(Lys)4-(Lys)2-Lys-Gly polypeptide compound.

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