Concanavalin A nano magnetic bead as well as preparation method and application thereof
By covalently coupling tartarin A with carboxylated superparamagnetic nano-scale magnetic beads, the problem of poor cell separation in the prior art was solved, and rapid , efficient cell isolation and glycosylated protein purification.
Patent Information
- Application Number
- CN202411445320.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-16
- Publication Date
- 2025-01-17
AI Technical Summary
In the prior art, ConA is mostly used for the enrichment and purification of glycosylated proteins, but the isolation effect of cells is poor, and a new ConA product is urgently needed to achieve rapid separation.
By covalently coupling tarton A with carboxylated superparamagnetic nano-scale magnetic beads, tarton A nano-magnetic beads are prepared and specific preparation methods include activation, coupling, blocking and washing steps to form Damolin A nanomagnetic beads with a mass ratio of 1:5 to 20.
The prepared Kalamol A nanomagnetic beads have low nonspecific adsorption, easy dispersion, and small particle size. They can quickly and efficiently isolate cells and purify glycosylated proteins, and are suitable for nuclease-targeted cleavage and release experiments.
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Figure CN119318943A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of immunodiagnosis, and in particular relates to concanavalin A nanomagnetic beads and a preparation method and application thereof. Background Art
[0002] Concanavalin A (ConA) is a carbohydrate-binding protein, also known as concanavalin A, concanavalin A, concanavalin A, and concanavalin A. ConA is a tetrameric globulin with a molecular weight of 102,000. Each subunit contains 237 amino acid residues and a molecular weight of 25,500. It binds to a Ca 2+ and a Mn 2+ , containing a sugar binding site. 2+ and Mn 2+ When present, ConA can bind to α-D-glucose or α-D-mannose in glycoproteins, glycolipids, and polysaccharides.
[0003] ConA is the first commercial lectin and is widely used in biochemistry to identify glycoproteins or other sugar-containing molecules. ConA is used in lectin affinity chromatography to purify biomacromolecules. ConA interacts with receptors for various mannose carbohydrates (serum and membrane glycoproteins). ConA causes strong agglutination of erythrocytes, is not blood type specific, and normal cells will reagglutinate after trypsin digestion. ConA is a mitogen for lymphocytes, mainly used to induce mitotic activity of T lymphocytes and increase the synthesis of cellular metabolites. ConA interacts with many bacteria, such as Escherichia coli, Dictyostelium and Bacillus subtilis. ConA is generally believed to be involved in the interaction between α-mannose oligosaccharides on the surface of HIV virus and human T lymphocytes.
[0004] In the prior art, ConA is mostly used for the enrichment and purification of molecules such as glycosylated proteins, but has poor separation effects on cells. Therefore, there is an urgent need to provide a new ConA product for rapid cell separation. Summary of the invention
[0005] In view of this, the object of the present invention is to provide a method for preparing concanavalin A nanomagnetic beads and application thereof.
[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0007] The present invention provides a concanavalin A nanomagnetic bead, which is formed by covalently coupling concanavalin A and carboxylated superparamagnetic nanoscale magnetic beads;
[0008] The mass ratio of the concanavalin A to the carboxylated superparamagnetic nano-scale magnetic beads is 1:5-20.
[0009] Preferably, the particle size of the carboxylated superparamagnetic nano-scale magnetic beads is 300 to 2000 nm.
[0010] The present invention also provides a method for preparing the Concanavalin A nanomagnetic beads, comprising the following steps:
[0011] 1) After washing the carboxylated superparamagnetic nano-scale magnetic beads, the beads are mixed with a 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution and an N-hydroxy-succinimide solution for activation to obtain activated carboxylated superparamagnetic nano-scale magnetic beads;
[0012] 2) The activated carboxylated superparamagnetic nano-scale magnetic beads are coupled to concanavalin A, blocked with 1× phosphate buffer containing bovine serum albumin, washed, and a preservation solution is added to obtain concanavalin A nano-magnetic beads.
[0013] Preferably, the washing in step 1) adopts 1×2-morpholineethanesulfonic acid buffer solution, wherein the 1×MEST solution contains Tween 20 with a volume concentration of 0.005-0.015%; the washing times are 1-3 times;
[0014] The mass volume ratio of the carboxylated superparamagnetic nano-scale magnetic beads and the 1×MEST solution is 5-10 mg:1 mL.
[0015] Preferably, the concentrations of the EDC solution and the NHS solution in step 1) are 5 to 15 mg / mL respectively;
[0016] The activation temperature is 22-27° C., and the activation time is 20-40 minutes.
[0017] Preferably, the coupling temperature in step 2) is 22-27° C. and the coupling time is 1-3 hours.
[0018] Preferably, in step 2), the mass volume fraction of BSA in the PBST solution is 0.5-1.5%, the blocking temperature is 22-27° C., and the blocking time is 0.5-2 hours.
[0019] Preferably, the washing in step 2) adopts 1×PBST solution, wherein the 1×PBST solution contains Tween 20 with a volume concentration of 0.005-0.015%, and the washing is performed 2-4 times.
[0020] Preferably, the preservation solution in step 2) is a 1×PBS solution containing 0.08-0.12% by mass volume fraction of bovine serum albumin and 0.08-0.12% by volume fraction of liquid biological preservative Proclin 300.
[0021] The present invention also provides the use of the Concanavalin A nanomagnetic beads or the Concanavalin A nanomagnetic beads prepared by the preparation method in cell separation, glycosylated protein purification or nuclease targeted cutting and release experiments.
[0022] Compared with the prior art, the present invention has the following beneficial effects:
[0023] The invention prepares concanavalin A nanomagnetic beads by activating and coupling carboxyl magnetic beads of different particle sizes to concanavalin A. The concanavalin A nanomagnetic beads prepared by the invention have the characteristics of low non-specific adsorption, easy dispersion, small particle size, and expression of concanavalin A, and can be used for rapid cell separation, rapid purification of glycosylated proteins, or for nuclease targeted cutting and release experiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 To evaluate the precipitation effect of concanavalin A (ConA) nanomagnetic beads using Hela cells (where A is a photo of Hela cells precipitated by ConA nanomagnetic beads taken under an inverted optical microscope, and B is a statistical graph of the number of Hela cells precipitated by ConA nanomagnetic beads);
[0025] Figure 2 To evaluate the precipitation effect of concanavalin A (ConA) nanomagnetic beads using Jurkat cells (where A is a photograph of Jurkat cells precipitated by ConA nanomagnetic beads taken under an inverted optical microscope, and B is a statistical diagram of the number of Jurkat cells precipitated by ConA nanomagnetic beads);
[0026] Figure 3 To evaluate the precipitation effect of concanavalin A (ConA) nanomagnetic beads using HCT116 cells (where A is a photograph of HCT116 cells precipitated by ConA nanomagnetic beads taken under an inverted optical microscope, and B is a statistical chart of the number of HCT116 cells precipitated by ConA nanomagnetic beads);
[0027] Figure 4 The comparison results of the precipitation effect of the present invention and the existing products are evaluated using Hela cells (wherein A is a photo of Hela cells precipitated by magnetic beads of different brands taken under an inverted optical microscope, and B is a statistical chart of the number of Hela cells precipitated by magnetic beads of different brands). DETAILED DESCRIPTION
[0028] The invention provides concanavalin A (ConA) nanomagnetic beads, which are formed by covalently coupling ConA and carboxylated superparamagnetic nanoscale magnetic beads; the mass ratio of the ConA to the carboxylated superparamagnetic nanoscale magnetic beads is preferably 1:5-20, and more preferably 1:10-15.
[0029] In the present invention, the particle size of the carboxylated superparamagnetic nano-scale magnetic beads is preferably 300 to 2000 nm, and more preferably 500 to 1500 nm.
[0030] The present invention also provides a method for preparing the ConA nanomagnetic beads, comprising the following steps:
[0031] 1) After washing the carboxylated superparamagnetic nano-scale magnetic beads, the beads are mixed with an EDC solution and an NHS solution for activation to obtain activated carboxylated superparamagnetic nano-scale magnetic beads;
[0032] 2) The activated carboxylated superparamagnetic nano-scale magnetic beads are coupled to ConA, blocked with a PBST solution containing BSA, washed, and a preservation solution is added to obtain ConA nano-magnetic beads.
[0033] In the present invention, after washing the carboxylated superparamagnetic nano-scale magnetic beads, the beads are mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) solution and N-hydroxy-succinimide (NHS) solution for activation to obtain activated carboxylated superparamagnetic nano-scale magnetic beads. The washing adopts 1×2-morpholineethanesulfonic acid buffer (1×MEST) solution, the 1×MEST solution contains Tween 20 with a volume concentration of 0.005-0.015%, and the volume concentration of Tween 20 is preferably 0.008-0.012%; the number of washings is preferably 1-3 times, more preferably 2 times; the concentrations of the EDC solution and the NHS solution are preferably 5-15 mg / mL, more preferably 8-12 mg / mL, respectively; the activation temperature is preferably 22-27°C, more preferably 22-27°C; the activation time is preferably 20-40 min, more preferably 25-35 min.
[0034] In the present invention, activated carboxylated superparamagnetic nano-scale magnetic beads are coupled to ConA, blocked with a 1× phosphate buffered saline (PBST) solution containing bovine serum albumin (BSA), washed, and a preservation solution is added to obtain concanavalin A nano-magnetic beads. The coupling temperature is preferably 22 to 27°C, more preferably 23 to 26°C; the coupling time is preferably 1 to 3 hours, more preferably 1.5 to 2.5 hours; the mass volume fraction of BSA in the PBST solution is preferably 0.5 to 1.5%, more preferably 0.8 to 1.2%; the blocking temperature is preferably 22 to 27°C, more preferably 23 to 26°C; the blocking time is preferably 0.5 to 2 hours, more preferably 0.8 to 1.5 hours; the washing adopts a 1×PBST solution, and the 1×PBST solution contains a volume concentration of 0.005 ~0.015% Tween 20, the volume concentration of Tween 20 is preferably 0.008~0.012%; the number of washings is preferably 2~4 times, and more preferably 3 times; the preservation solution is a 1×PBS solution containing 0.08~0.12% mass volume fraction of bovine serum albumin and 0.08~0.12% volume fraction of liquid biological preservative Proclin300; the mass volume fraction of bovine serum albumin is preferably 0.09~0.11%; the volume fraction of the liquid biological preservative Proclin300 is preferably 0.09~0.11%.
[0035] The present invention also provides the use of the ConA nanomagnetic beads or the ConA nanomagnetic beads prepared by the preparation method in cell separation, purification of glycosylated proteins or nuclease targeted cutting and release (CUT&RUN) experiments. The ConA nanomagnetic beads can quickly, efficiently, sensitively and specifically bind to molecules with glycosylation modification such as glycoproteins, glycolipids, polysaccharides, etc., and can be very conveniently used to separate cells with the help of magnetic separation equipment such as magnetic racks, or to separate glycosylation-modified molecules such as glycoproteins in cells, tissue lysates or serum, and can also be used for nuclease targeted cutting and release of chromatin.
[0036] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0037] The main reagents and consumables in the experimental process of the present invention are as follows:
[0038] Carboxyl magnetic beads, Suzhou Beaver Biotechnology, catalog number: 70106-5
[0039] MES Solarbio, Article No.: M8010-100 g
[0040] EDC Beyotime, SKU: ST1299-25g
[0041] NHS Solarbio, Part Number: H8220
[0042] Concanavalin A was stored in this laboratory.
[0043] Tween 20 Beyotime, item number: ST1726-500 ml
[0044] Bovine serum albumin Solarbio, Product number: A8020-100 g
[0045] PBS Hyclone, Catalog Number: SH30256
[0046] Proclin300 Aladdin, item number: P406359
[0047] Magnetic separator, Suzhou Beaver Biological, catalog number: 60201
[0048] BeyoMag TM ConAMag BeadsBeijing Bio-Technology Co., Ltd.
[0049] ConAMag BeadsShanghai Lingyin Biotechnology Co., Ltd.
[0050] VAHTS ConABeads ProNanjing Novogene Biotech Co., Ltd.
[0051] CST ConAMag BeadsCST USA
[0052] Example 1
[0053] Take 100μl of 10mg / ml 300nm carboxylated polymer magnetic beads in a 1.5ml centrifuge tube, and then remove the supernatant by magnetic separation. Then, wash twice with 200μl MEST solution (100mM, pH5.0; containing 0.01% Tween 20). After removing the supernatant, quickly add 200μl 20mg / ml EDC solution and 200μl 20mg / ml NHS solution to the magnetic beads, mix well, and activate at 25℃ for 30min.
[0054] The activated carboxylated magnetic beads were separated by magnetic separation, and the supernatant was removed. Then, 200 μg of ConA (adjusted to 100 μl with 1×PBS) was added, mixed gently, and coupled at 25°C for 2 hours. After the coupling, the supernatant was removed, and 200 μl of PBST solution containing 1% bovine serum albumin (1×PBS, pH7.2; containing 0.01% Tween 20) was added, and blocked at 25°C for 1 hour. After the blocking was completed, the supernatant was removed, and the beads were washed 3 times with 200 μl of 1×PBST (containing 0.01% Tween 20 by volume), and finally 100 μl of preservation solution (1×PBS containing 0.1% bovine serum albumin and 0.1% Proclin300) was added to prepare ConA magnetic beads, which were stored at 4°C.
[0055] Example 2
[0056] Take 200μl of 10mg / ml 500nm carboxylated polymer magnetic beads in a 1.5ml centrifuge tube, and then remove the supernatant by magnetic separation. Then, wash once with 200μl MEST solution (100mM, pH5.0; containing 0.005% Tween 20). After removing the supernatant, quickly add 200μl of freshly prepared 5mg / ml EDC solution and 200μl of 5mg / ml NHS solution to the magnetic beads, mix well, and activate at 22℃ for 20min.
[0057] The activated carboxylated magnetic beads were separated by magnetic separation, and the supernatant was removed. Then, 200 μg of ConA (adjusted to 100 μl with 1×PBS) was added, mixed gently, and coupled at 22°C for 1 hour. After the coupling, the supernatant was removed, and 200 μl of PBST solution containing 0.5% bovine serum albumin (1×PBS, pH7.2; containing 0.01% Tween 20) was added, and blocked at 22°C for 0.5 hour. After the blocking was completed, the supernatant was removed, and the beads were washed twice with 200 μl of 1×PBST (containing 0.005% Tween 20 by volume), and finally 100 μl of preservation solution (1×PBS containing 0.08% bovine serum albumin and 0.08% Proclin300) was added to prepare ConA magnetic beads, which were stored at 4°C.
[0058] Example 3
[0059] Take 400μl 10mg / ml 2μm carboxylated polymer magnetic beads in a 1.5ml centrifuge tube, and then remove the supernatant by magnetic separation. Then, wash 3 times with 200μl MEST solution (100mM, pH5.0; containing 0.015% Tween 20). After removing the supernatant, quickly add 200μl 15mg / ml EDC solution and 200μl 15mg / ml NHS solution to the magnetic beads, mix well, and activate at 27℃ for 40min.
[0060] The activated carboxylated magnetic beads were separated by magnetic separation, and the supernatant was removed. Then, 200 μg of ConA (adjusted to 100 μl with 1×PBS) was added, mixed gently, and coupled at 27°C for 3 hours. After the coupling, the supernatant was removed, and 200 μl of PBST solution containing 1.5% bovine serum albumin (1×PBS, pH7.2; containing 0.01% Tween 20) was added, and blocked at 27°C for 2 hours. After the blocking was completed, the supernatant was removed, and the beads were washed 4 times with 200 μl of 1×PBST (containing 0.015% Tween 20 by volume), and finally 100 μl of preservation solution (1×PBS containing 0.12% bovine serum albumin and 0.12% Proclin300) was added to prepare ConA magnetic beads, which were stored at 4°C.
[0061] Experimental Example 1
[0062] Take 300nm, 500nm, and 2μm carboxylated polymer magnetic beads, use ConA and bovine serum albumin (negative control) as conjugates, respectively, and prepare 300nm carboxyl magnetic beads coupled to bovine serum albumin reference substance (300nm Mag COOH Beads), 300nm carboxyl magnetic beads coupled to ConA test substance (ConA-conjugated 300nm Mag COOH Beads), 500nm carboxyl magnetic beads coupled to bovine serum albumin reference substance (500nm Mag COOH Beads), 500nm carboxyl magnetic beads coupled to ConA test substance (ConA-conjugated 500nm Mag COOH Beads), 2μm carboxyl magnetic beads coupled to bovine serum albumin reference substance (2μm Mag COOH Beads), and 2μm carboxyl magnetic beads coupled to ConA test substance (ConA-conjugated 2μm Mag COOH Beads) according to the method of Example 1.
[0063] Remove the culture medium from HeLa and HCT116 cells, wash the cells with Washing Buffer I (20mM Hepes (pH7.5), 150mM NaCl), and then add 1-2ml trypsin to digest the cells; after digestion, add serum-containing culture medium to terminate digestion, centrifuge at 800×g for 5min at 4℃, remove the supernatant; then add Washing Buffer I to resuspend and wash, and then centrifuge at 800×g for 5min at 4℃ to collect the cells. For Jurkat cells, collect the cells by centrifugation at 800×g for 5min at 4℃, remove the supernatant, resuspend and wash with Washing Buffer I, and centrifuge at 800×g for 5min at 4℃ to collect the cells. Then add 5ml Binding Buffer (20mM Hepes (pH7.4), 150mM NaCl, 1mM MgCl2, 1mM MnCl2, 1mM CaCl2) to resuspend the cells, and adjust the number of cells in the sample to 20,000-200,000 by counting. According to the ratio of adding 10μl of ConA magnetic bead suspension to every 500μl sample, take 50μl ConA magnetic beads into a clean centrifuge tube, add 10 times the volume of Binding Buffer, and wash twice (use a magnetic separator to remove the supernatant). According to the initial volume of 50μl ConA magnetic beads, resuspend the ConA magnetic beads with 50μl Binding Buffer to obtain activated ConA magnetic beads. Add an appropriate amount of ConA magnetic beads to the sample, gently resuspend, place on a rotating mixer, and incubate at room temperature for 30min (or incubate overnight at 4℃). Place the centrifuge tube containing the sample on a magnetic stand for separation and remove the supernatant. Take 0.5 ml of Washing Buffer II (20 mM Hepes (pH 7.4), 150 mM NaCl, 1 mM MgCl2, 1 mM MnCl2, 1 mM CaCl2, 0.1% Tween-20) and add it to the separated magnetic beads. Gently resuspend the magnetic beads and wash them on a rotating mixer for 5 minutes. Then, separate them using a magnetic stand and remove the supernatant. Repeat the washing 3 times. Then take pictures and count them under a microscope.
[0064] Experimental results: Figures 1 to 3 As shown in the figure, ConA-conjugated 300nm MagCOOH Beads can effectively precipitate HeLa, HCT116 and Jurkat cells compared to ConA-conjugated 500nm and 2μm Mag COOH Beads.
[0065] Experimental Example 2
[0066] According to the method of Experimental Example 1, the ConA-conjugated 300nm Mag COOHBeads constructed by the present invention were compared with the commercially available product BeyoMag TM ConA Mag Beads, The difference in effects between ConAMag Beads, VAHTSConABeads Pro and CST ConAMag Beads.
[0067] Experimental results: Figure 4 As shown. The effect of ConA-conjugated 300nm Mag COOH Beads in precipitating HeLa cells is significantly better than BeyoMag TM ConA Mag Beads and ConAMag Beads; comparable to VAHTSConABeads Pro and CST ConAMag Beads, with no significant difference.
[0068] It can be seen from the above embodiments and experimental examples that the ConA nanomagnetic beads prepared by the present invention have the characteristics of low nonspecific adsorption, easy dispersion, small particle size, expression of ConA, etc., and can quickly and effectively separate cells.
[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A concanavalin A nanomagnetic bead, characterized in that: It is composed of concanavalin A and carboxylated superparamagnetic nano-sized magnetic beads covalently coupled; The mass ratio of the concanavalin A to the carboxylated superparamagnetic nano-scale magnetic beads is 1:5-20.
2. The Concanavalin A nanoparticle magnetic bead according to claim 1, characterized in that The particle size of the carboxylated superparamagnetic nano-scale magnetic beads is 300-2000nm.
3. The method for preparing the Concanavalin A nano-magnetic beads according to claim 1 or 2, characterized in that: The following steps are involved: 1) After washing the carboxylated superparamagnetic nano-scale magnetic beads, the beads are mixed with a 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution and an N-hydroxy-succinimide solution for activation to obtain activated carboxylated superparamagnetic nano-scale magnetic beads; 2) The activated carboxylated superparamagnetic nano-scale magnetic beads are coupled to concanavalin A, blocked with 1× phosphate buffer containing bovine serum albumin, washed, and a preservation solution is added to obtain concanavalin A nano-magnetic beads.
4. The preparation method according to claim 3, characterized in that: Step 1) The washing is performed using 1×2-morpholineethanesulfonic acid buffer, wherein the 1×MEST solution contains Tween 20 with a volume concentration of 0.005 to 0.015%; the washing is performed 1 to 3 times; The mass volume ratio of the carboxylated superparamagnetic nano-scale magnetic beads and the 1×MEST solution is 5-10 mg:1 mL.
5. The preparation method according to claim 3, characterized in that: Step 1) The concentrations of the EDC solution and the NHS solution are 5-15 mg / mL respectively; The activation temperature is 22-27° C., and the activation time is 20-40 minutes.
6. The preparation method according to claim 3, characterized in that: Step 2) The coupling temperature is 22-27° C.; the coupling time is 1-3 hours.
7. The preparation method according to claim 3, characterized in that: Step 2) The mass volume fraction of BSA in the 1×PBST solution is 0.5-1.5%, the blocking temperature is 22-27° C., and the blocking time is 0.5-2 hours.
8. The preparation method according to claim 3, characterized in that: Step 2) The washing is performed using 1×PBST solution, which contains Tween 20 with a volume concentration of 0.005-0.015%, and the washing is performed 2-4 times.
9. The preparation method according to claim 3, characterized in that: Step 2) The preservation solution is a 1×PBS solution containing 0.08-0.12% by mass volume fraction of bovine serum albumin and 0.08-0.12% by volume fraction of liquid biological preservative Proclin 300.
10. Use of the Concanavalin A nanomagnetic beads according to claim 1 or 2 or the Concanavalin A nanomagnetic beads prepared by the preparation method according to any one of claims 3 to 9 in separating cells, purifying glycosylated proteins or in nuclease targeted cleavage and release experiments.